Radionuclide metal N2 S2 chelates substituted with glucose and biotin moieties

ABSTRACT

Chelating compounds of specified structure are useful for radiolabeling targeting proteins such as antibodies as well as proteinaceous and non-proteinaceous ligands and anti-ligands. The radiolabeled antibodies, ligands or anti-ligands, or catabolites thereof, demonstrate improved biodistribution properties, including reduced localization within the intestines.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/US94/07733, filed Jul. 12, 1994, whichis a continuation of U.S. Ser. No. 08/090,421, filed Jul. 12, 1993, nowabandoned; which is a continuation-in-part of U.S. Ser. No. 07/973,048,filed Nov. 6, 1992, U.S. Pat. No. 5,250,666; which is a divisional ofU.S. Ser. No. 07/577,959, filed Sep. 5, 1990, U.S. Pat. No. 5,164,176;which is a continuation-in-part of U.S. Ser. No. 07/367,502, filed onJun. 16, 1989, now abandoned.

BACKGROUND

Radiolabeled antibodies are used in a variety of diagnostic andtherapeutic medical procedures. The increased specificity of monoclonalantibodies, compared to polyclonal antibodies, makes them even moreuseful for delivering diagnostic or therapeutic agents such asradioisotopes to desired target sites in vivo. A monoclonal antibodyspecific for a desired type of target cells such as tumor cells may beused to deliver a therapeutic radionuclide attached to the antibody tothe target cells, thereby causing the eradication of the undesiredtarget cells. Alternatively, a monoclonal antibody having adiagnostically effective radionuclide attached thereto may beadministered, whereupon the radiolabeled antibody localizes on thetarget tissue. Conventional diagnostic procedures then may be used todetect the presence of the target sites within the patient. In contrastto such "chelate-labeled antibody" procedures, pretargeting approachesmay be used to achieve therapeutic or diagnostic goals, whichpretargeting approaches involve the interaction of two members of a highaffinity binding pair such as a ligand-anti-ligand binding pair.

One method for radiolabeling proteins such as antibodies as well asproteinaceous and non-proteinaceous binding pair members involvesattachment of radionuclide metal chelates to the proteins or bindingpair members. Chelates having a variety of chemical structures have beendeveloped for this purpose. The usefulness of such chelates is dependentupon a number of factors such as the stability of radionuclide bindingwithin the chelate and the reactivity of the a chelate with the desiredprotein or binding pair member. The efficiency of radiolabeling of thechelating compound to produce the desired radionuclide metal chelatealso is important. Another consideration is the biodistribution of theradiolabeled antibody or binding pair member and catabolites thereof invivo. Localization in non-target tissues limits the total dosage of atherapeutic radiolabeled antibody or binding pair member that can beadministered, thereby decreasing the therapeutic effect. In diagnosticprocedures, localization in non-target tissues may cause undesirablebackground and/or result in misdiagnosis. The need remains forimprovement in these and other characteristics of radionuclide metalchelate compounds used for radiolabeling of proteins such as antibodies.The use of pretargeting approaches diminishes non-target tissuelocalization of radiolabel; however, the need remains for improvement inmolecules incorporating chelates and binding pair members ofproteinaceous or non-proteinaceous structure.

SUMMARY OF THE INVENTION

The present invention provides a compound of the formula: ##STR1##wherein: each R independently represents ═O, H₂, a lower alkyl group,--(CH₂)_(n) -- COOH, --(CH₂)_(n) --CO-saccharide or saccharidederivative, or --(CH₂)_(n) --NH-saccharide or saccharide derivative, orR₁ --Z;

n is 0 to about 3;

R₁ represents a lower alkyl or substituted lower alkyl group;

Z represents a protein conjugation group, a ligand conjugation group, ananti-ligand conjugation group or a targeting protein, ligand oranti-ligand; or a ligand-linker moiety or an anti-ligand-linker moietywherein the linker moiety is derived from a ligand or anti-ligandconjugation group;

each R₂ independently represents H₂, a lower alkyl group, --(CH₂)_(n) --COOH, --(CH₂)_(n) --CO-saccharide or saccharide derivative, or--(CH₂)_(n) --NH-saccharide or saccharide derivative, or R₁ --Z;

each m is 0 or 1, with at most one m=1;

each T represents a sulfur protecting group; and

the compound comprises at least one (CH₂)_(n) --COOH substituent or one--(CH₂)_(n) --CO-saccharide or saccharide derivative or --(CH₂)_(n)--NH-saccharide or saccharide derivative substituent and one --R₁ --Zsubstituent.

The present invention also provides radionuclide metal chelate compoundsof the formula: ##STR2## wherein: M represents a radionuclide metal oroxide thereof and the other symbols are as described above.

These compounds comprise a targeting protein such as an antibody, or aconjugation group for attachment of the compound to a targeting protein.Alternatively, the compounds include a ligand or an anti-ligand or aconjugation group for the attachment of the compound to a ligand or toan anti-ligand. The chelating compound may be attached to a targetingprotein, ligand or anti-ligand and subsequently radiolabeled.Alternatively, the radionuclide metal chelate compound may be preparedand then attached to a targeting protein, ligand or anti-ligand. Theresulting radiolabeled targeting proteins, ligands or anti-ligands areuseful in diagnostic and therapeutic medical procedures. An example of atargeting protein is a monoclonal antibody that binds to cancer cells.An example of a ligand is biotin, with the complementary anti-ligandthereof being avidin or streptavidin, wherein biotin and avidin orstreptavidin together form a ligand-anti-ligand binding pair.

Some additional compounds of the present invention incorporate an estercleavable R₁ moiety exhibiting, for example, ester and/or amidefunctionalities. An example of a chelate-biotin conjugate of this aspectof the present invention, involving a succinate mono-ester mono-amide,is shown below: ##STR3## wherein X is H or COOH.

The carboxylic acid substituent(s) on the compounds of the presentinvention are believed to assist in chelation of a radionuclide and tocontribute to improved biodistribution properties of catabolites of theradiolabeled targeting proteins, ligands or anti-ligands. Reducedlocalization of radioactivity within the intestines is achieved usingthe radiolabeled targeting proteins, ligands or anti-ligands of thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-7 depict chemical synthesis procedures that may be used toprepare certain chelating compounds of the present invention.

FIG. 8 depicts the tumor uptake profile of NR-LU-10 streptavidinconjugate (LU-10-StrAv) in comparison to a control profile of nativeNR-LU-10 whole antibody.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention, it may be helpful to set forthdefinitions of certain terms to be used within the disclosure.

Targeting moiety: A molecule that binds to a defined population ofcells. The targeting moiety may bind a receptor, an oligonucleotide, anenzymatic substrate, an antigenic determinant, or other binding sitepresent on or in the target cell population. Targeting moieties that areproteins are referred to herein as "targeting proteins." Antibody isused throughout the specification as a prototypical example of atargeting moiety and a targeting protein. Tumor is used as aprototypical example of a target in describing the present invention.

Ligand/anti-ligand pair: A complementary/anticomplementary set ofmolecules that demonstrate specific binding, generally of relativelyhigh affinity. Exemplary ligand/anti-ligand pairs include zinc fingerprotein/dsDNA fragment, hapten/antibody, lectin/carbohydrate,ligand/receptor, and biotin/avidin. Biotin/avidin is used throughout thespecification as a prototypical example of a ligand/anti-ligand pair.

Anti-ligand: As defined herein, an "anti-ligand" demonstrates highaffinity, and preferably, multivalent binding of the complementaryligand. Preferably, the anti-ligand is large enough to avoid rapid renalclearance, and contains sufficient multivalency to accomplishcrosslinking and aggregation of targeting moiety-ligand conjugates.Univalent anti-ligands are also contemplated by the present invention.Anti-ligands of the present invention may exhibit or be derivitized toexhibit structural features that direct the uptake thereof, e.g.,galactose residues that direct liver uptake. Avidin and streptavidin areused herein as prototypical anti-ligands.

Avidin and Streptavidin: As defined herein, both of the terms "avidin"and "streptavidin" include avidin, streptavidin and derivatives andanalogs thereof that are capable of high affinity, multivalent orunivalent binding of biotin.

Ligand: As defined herein, a "ligand" is a relatively small, solublemolecule that exhibits rapid serum, blood and/or whole body clearancewhen administered intravenously in an animal or human. Biotin is used asthe prototypical ligand.

Pretargeting: As defined herein, pretargeting involves target sitelocalization of a targeting moiety that is conjugated with one member ofa ligand/anti-ligand pair; after a time period sufficient for optimaltarget-to-non-target accumulation of this targeting moiety conjugate,active agent conjugated to the opposite member of the ligand/anti-ligandpair is administered and is bound (directly or indirectly) to thetargeting moiety conjugate at the target site (two-step pretargeting).Three-step and other related methods described herein are alsoencompassed.

Linker Moiety: A moiety that is a portion of a protein, ligand oranti-ligand conjugation group that remains part of the structure of aprotein-chelate, ligand-chelate or anti-ligand-chelate conjugatefollowing the conjugation step. For example, the linker moiety of anactive ester chelate derivative includes, for example, a carbonyl(--CO--) moiety.

The present invention provides chelating compounds and radionuclidemetal chelate compounds prepared therefrom, as well as radiolabeledproteins, ligands or anti-ligands having the chelates attached thereto.The radionuclide metal chelates of the present invention are attached totargeting proteins such as antibodies to form radiolabeled targetingproteins having diagnostic or therapeutic use. The compounds eachcomprise a targeting protein or a protein conjugation group forattachment of the compound to a targeting protein. Alternatively, theradionuclide metal chelates of the present invention are attached toligands or anti-ligands to form radiolabeled ligands or anti-ligandshaving diagnostic or therapeutic use. Such compounds include a ligand oranti-ligand conjugation group to facilitate attachment of the compoundto a ligand or anti-ligand. The compounds also comprise at least onecarboxylic acid substituent. The good radiolabeling yields (i.e.,chelate formation) achieved with these compounds are believed to beattributable, at least in part, to the presence of the carboxylic acidsubstituent(s). The improved biodistribution properties of theradiolabeled proteins of the invention also are believed to be at leastin part attributable to the carboxylic acid substituent(s) on thechelate.

Provided by the present invention are chelating compounds of thefollowing formula: ##STR4## wherein: each R independently represents ═O,H₂, a lower alkyl group, --(CH₂)_(n) --COOH, --(CH₂)_(n) --CO-saccharideor saccharide derivative, or --(CH₂)_(n) --NH-saccharide or saccharidederivative, or R₁ --Z;

n is 0 to 3;

R₁ represents a lower alkyl or substituted lower alkyl group;

Z represents a protein conjugation group, a ligand conjugation group, ananti-ligand conjugation group or a targeting protein, ligand oranti-ligand; or a ligand-linker moiety or an anti-ligand-linker moietywherein the linker moiety is derived from a ligand or an anti-ligandconjugation group;

each R₂ independently represents H₂, a lower alkyl group, --(CH₂)_(n)--COOH, --(CH₂)_(n) --CO-saccharide or saccharide derivative, or--(CH₂)_(n) --NH-saccharide or saccharide derivative, or R₁ --Z;

each m is 0 or 1, with at most one m=1;

each T represents a sulfur protecting group; and

the compound comprises at least one --(CH₂)_(n) --COOH substituent--(CH₂)_(n) --CO-saccharide or saccharide derivative, or --(CH₂)_(n)--NH-saccharide or saccharide derivative substituent and one --R₁ --Zsubstituent.

The above presented chelating compounds are radiolabeled to form thecorresponding radionuclide metal chelates of the following formula:##STR5## wherein: M represents a radionuclide metal or an oxide thereofand all the other symbols are as described above.

Some additional compounds of the present invention incorporate estercleavable R₁ moieties, incorporating, for example, ester and/oramide-containing R₁ groups. An example of such compounds of the presentinvention employing a cleavable succinate mono-ester mono-amide linkagehas the formula shown below: ##STR6## wherein X is H or COOH. Theadvantage of an ester cleavable R₁ group is a reduction in non-targetcell retention. Also, ester functionalities often improve watersolubility and overall polarity of small molecules. Preparation ofcompounds having ester cleavable linkers is discussed in the Examplesset forth below.

A protein conjugation group is a chemically reactive functional groupthat will react with a protein under conditions that do not denature orotherwise adversely affect the protein. The protein conjugation grouptherefore is sufficiently reactive with a functional group on a proteinso that the reaction can be conducted in substantially aqueous solutionsand does not have to be forced, e.g. by heating to high temperatures,which may denature the protein. Examples of suitable protein conjugationgroups include but are not limited to active esters, isothiocyanates,amines, hydrazines, thiols, and maleimides. Among the preferred activeesters are thiophenyl ester, 2,3,5,6-tetrafluorophenyl ester, and2,3,5,6-tetrafluorothiophenyl ester. The preferred active esters maycomprise a group that enhances water solubility, at the para (i.e., 4)position on the phenyl ring. Examples of such groups are CO₂ H, SO₃ ⁻,PO₃ ²⁻ and OPO₃ ²⁻, and O(CH₂ CH₂ O)_(n) CH₃ groups.

A ligand or anti-ligand conjugation group is a chemically reactivefunctional group that will react with a ligand or anti-ligand underconditions that do not adversely affect the ligand or anti-ligand,including the capacity of the ligand or anti-ligand to bind to itscomplementary binding pair member. Ligand or anti-ligand conjugationgroups therefore are sufficiently reactive with a functional group on aligand or anti-ligand so that the reaction can be conducted underrelatively mild reaction conditions including those described above forprotein-chelate conjugation. For proteinaceous ligands or anti-ligands,such as streptavidin, protein conjugation groups may correspond toligand or anti-ligand conjugation groups. Examples of suitable ligand oranti-ligand conjugation groups therefore include, but are not limitedto, active esters, isothiocyanates, amines, hydrazines, thiols, andmaleimides. Among the preferred active esters are thiophenyl ester,2,3,5,6-tetrafluorophenyl ester, and 2,3,5,6-tetrafluorothiophenylester. The preferred active esters may comprise a group that enhanceswater solubility, at the para (i.e., 4) position on the phenyl ring.Examples of such groups are CO₂ H, SO₃ ⁻, PO₃ ²⁻ -, OPO₃ ²⁻, and O(CH₂CH₂ O)_(n) CH₃ groups.

For non-proteinaceous ligand or anti-ligand moieties, such as biotin,suitable conjugations groups are those functional groups that react witha ligand or anti-ligand functional group (e.g., a terminal carboxygroup) or a functional group which the ligand or anti-ligand has beenderivatized to contain (e.g., an alcohol or an amine group produced bythe reduction of a terminal carboxy moiety). As a result, conjugationgroups, such as those recited above, that are capable of reacting with--COOH, --OH or --NH₂ groups are useful conjugation groups for producingbiotin-chelate molecules of this aspect of the present invention.Exemplary biotin-COOH conjugation groups are amines, hydrazines,alcohols and the like. Exemplary biotin-OH conjugation groups aretosylates (Ts), active esters, halides and the like, with exemplarygroups being reactive with biotin-O-Ts including amines, hydrazines,thiols and the like. Exemplary biotin-NH₂ conjugation groups are activeesters, acyl chlorides, tosylates, isothiocyanates and the like.

The protein conjugation group, ligand conjugation group, or anti-ligandconjugation group (represented as Z in the above-presented formulas) isattached to the chelating compound core through the linkage representedas R₁. R₁ is a lower alkyl or substituted lower alkyl group. By "loweralkyl" is meant an alkyl group of preferably one to four carbon atoms.Most preferably, R₁ is a methylene chain comprising from two to threecarbon atoms. The lower alkyl group may be substituted with hetero atomssuch as oxygen or nitrogen atoms. When the protein conjugation group,ligand conjugation group, or anti-ligand conjugation group is a primaryamine, the R₁ linkage comprises a methylene group immediately adjacentto the terminal primary amine protein conjugation group.

The "targeting moiety" of the present invention binds to a definedtarget cell population, such as tumor cells. Preferred targetingmoieties useful in this regard include antibody and antibody fragments,proteinaceous or non-proteinaceous ligands or anti-ligands, peptides,and hormones. Proteins corresponding to known cell surface receptors(including low density lipoproteins, transferrin and insulin),fibrinolytic enzymes, anti-HER2, platelet binding proteins such asannexins, and biological response modifiers (including interleukin,interferon, erythropoietin and colony-stimulating factor) are alsopreferred targeting moieties. Also, anti-EGF receptor antibodies, whichinternalize following binding to the receptor and traffic to the nucleusto an extent, are preferred targeting moieties for use in the presentinvention to facilitate delivery of Auger emitters and nucleus bindingdrugs to target cell nuclei. Oligonucleotides, e.g., antisenseoligonucleotides that are complementary to portions of target cellnucleic acids (DNA or RNA), are also useful as targeting moieties in thepractice of the present invention. Oligonucleotides binding to cellsurfaces are also useful. Analogs of the above-listed targeting moietiesthat retain the capacity to bind to a defined target cell population mayalso be used within the claimed invention. In addition, synthetictargeting moieties may be designed.

Functional equivalents of the aforementioned molecules are also usefulas targeting moieties of the present invention. One targeting moietyfunctional equivalent is a mimetic compound, an organic chemicalconstruct designed to mimic the proper configuration and/or orientationfor targeting moiety-target cell binding. Another targeting moietyfunctional equivalent is a short polypeptide designated as a "minimal"polypeptide, constructed using computer-assisted molecular modeling andmutants having altered binding affinity, which minimal polypeptidesexhibit the binding affinity of the targeting moiety.

The term "targeting protein" as used herein refers to proteins which arecapable of binding to a desired target site in vivo. The targetingprotein may bind to a receptor, substrate, antigenic determinant,complementary binding pair member or other binding site on a target cellor other target site. The targeting protein serves to deliver theradionuclide attached thereto to the desired target site in vivo.Examples of targeting proteins include, but are not limited to,antibodies and antibody fragments, proteinaceous ligands oranti-ligands, hormones, fibrinolytic enzymes, and biologic responsemodifiers. The term "targeting protein" includes proteins, polypeptides,and fragments thereof. In addition, other molecules that localize in adesired target site in vivo, although not strictly proteins, areincluded within the definition of the term "targeting proteins" as usedherein. For example, certain carbohydrates or glycoproteins may be usedin the present invention. The proteins may be modified, e.g., to producevariants and fragments thereof, as long as the desired biologicalproperty (i.e., the ability to bind to the target site) is retained. Theproteins may be modified by using various genetic engineering or proteinengineering techniques.

Among the preferred targeting proteins are antibodies, most preferablymonoclonal antibodies. A number of monoclonal antibodies that bind to aspecific type of cell have been developed, including monoclonalantibodies specific for tumor-associated antigens in humans. Among themany such monoclonal antibodies that may be used are anti-TAC, or otherinterleukin-2 receptor antibodies; 9.2.27 and NR-ML-05 to the 250kilodalton human melanoma-associated proteoglycan; and NR-LU-10 to apancarcinoma glycoprotein. The antibody employed in the presentinvention may be an intact (whole) molecule, a fragment thereof, or afunctional equivalent thereof. Examples of antibody fragments areF(ab')₂, Fab', Fab, and F_(v) fragments, which may be produced byconventional methods or by genetic or protein engineering.

Human monoclonal antibodies or "humanized" murine antibodies are alsouseful as targeting moieties in accordance with the present invention.For example, murine monoclonal antibody may be "humanized" bygenetically recombining the nucleotide sequence encoding the murine Fvregion (i.e., containing the antigen binding site) or thecomplementarity determining regions thereof with the nucleotide sequenceencoding at least a human constant domain region and an Fc region, e.g.,in a manner similar to that disclosed in European Patent ApplicationPublication No. 0,411,893 A3. Some additional murine residues may alsobe retained within the human variable region framework domains to ensureproper target site binding characteristics. Humanized targeting moietiesare recognized to decrease the immunoreactivity of the antibody orpolypeptide in the host recipient, permitting an increase in thehalf-life and a reduction in the possibility of adverse immunereactions.

Targeting proteins are rarely completely specific for a desired targetsite. Localization in non-target tissues may occur throughcross-reactivity or non-specific uptake, for example. In the case ofradiolabeled targeting proteins, such localization at non-target sitesmay result in decreased clarity of diagnostic images (due to theincreased "background") and misdiagnosis. Exposure of non-target tissuesto radiation also occurs, which is especially undesirable in therapeuticprocedures. The improved biodistribution properties of the radiolabeledtargeting proteins of the present invention are believed to beattributable to the effect of the chelate, most likely on thebiodistribution of catabolites of the radiolabeled proteins.

Ligands suitable for use within the present invention include biotin,haptens, lectins, epitopes, dsDNA fragments and analogs and derivativesthereof. Useful complementary anti-ligands include avidin (for biotin),carbohydrates (for lectins), antibody, fragments or analogs thereof,including mimetics (for haptens and epitopes) and zinc finger proteins(for dsDNA fragments). Preferred ligands and anti-ligands bind to eachother with an affinity of at least about k_(D) ≧10⁻⁹ M.

The chelating compounds of the present invention comprise two nitrogenand two sulfur donor atoms, and thus may be termed "N₂ S₂ " chelatingcompounds. The radiolabeled targeting proteins of the present inventionexhibit certain improved biodistribution properties compared totargeting proteins radiolabeled with certain other N₂ S₂ chelates. Mostnotably, localization of radiolabeled targeting proteins (or catabolitesthereof) within the intestines is reduced.

Targeting proteins radiolabeled with certain N₂ S₂ radionuclide metalchelates are described, for example, in European Patent ApplicationPublication Number 188,256. When the radiolabeled proteins of EP 188,256are administered in vivo, a percentage of the injected dosage of theradionuclide becomes localized within the intestines (i.e., becomes partof the intestinal contents, rather than binding to intestinal epithelialtissue per se). Although stable attachment of radionuclides toantibodies and effective localization thereof on target tumors has beenachieved using the EP 188,256 system, reduction of the intestinallocalization would be beneficial. A portion of the non-target-boundadministered radiolabeled proteins (e.g., antibodies or fragmentsthereof) most likely is first metabolized to produce radiolabeledcatabolites that subsequently enter the intestines, probably throughhepatobiliary excretion. When the chelate is attached to lysine residuesof the targeting protein, a major catabolite may be the lysine adduct ofthe chelate. Intestinal localization of radioactivity may be confusedwith (or obstruct) target sites in the abdominal area. For therapeuticprocedures, the dosage that can be safely administered is reduced whenintestinal localization occurs (due to exposure of normal tissues to theradiation). The therapeutic effect on the target sites therefore also isreduced.

As illustrated in the examples below, the biodistribution patterns invivo differ when targeting proteins (e.g., antibody fragments) areradiolabeled with a chelate of the present invention, compared toradiolabeling using certain other N₂ S₂ chelates. The advantage ofreduced intestinal localization is demonstrated for the radiolabeledtargeting proteins of the present invention. While not wishing to bebound by theory, it is believed that the carboxylic acid substituent(s)on the chelate confer the advantageous biodistribution properties oncatabolites of the radiolabeled protein (most likely lysine adducts ofthe chelate). The carboxylic acid substituent(s) on the compounds of thepresent invention increase the polarity, and therefore the watersolubility, of the compounds. The increased water solubility is believedto promote excretion of the catabolites by the kidneys, resulting inefficient elimination of the radioactive catabolites in the urine. Othersubstituents that enhance polarity (e.g., sulfate groups) may be used onthe chelating compounds, in addition to (or instead of) the COOHsubstituents.

Another advantage of the chelates of the present invention is thecomparatively good radiolabeling yields. The free carboxylic acidsubstituent(s) are believed to assist in the chelation of theradionuclide. Radiolabeled ligands and anti-ligands also exhibit thesefavorable biodistribution and chelation properties.

During radiolabeling, bonds form between the four donor atoms and theradionuclide metal to form the corresponding radionuclide metal chelate.Any suitable conventional sulfur protecting group(s) may be attached tothe sulfur donor atoms of the compounds of the present invention. Theprotecting groups should be removable, either prior to or during theradiolabeling reaction. The protecting groups attached to the two sulfurdonor atoms may be the same or different. Alternatively, a singleprotecting group, e.g. a thioacetal group, may protect both sulfur donoratoms. Among the preferred sulfur protecting groups are acetamidomethyland hemithioacetal protecting groups, which are displacable from thechelating compound during the radiolabeling reaction. Preferably, atleast one sulfur protecting group is a hemithioacetal group, and at mostone sulfur protecting group is an acetamidomethyl group.

An acetamidomethyl sulfur-protecting group is represented by thefollowing formula, wherein the sulfur atom shown is a sulfur donor atomof the chelating compound: ##STR7##

The acetamidomethyl group is displaced from the chelating compoundduring radiolabeling conducted at about 50° C. in a reaction mixturehaving a pH of about 3 to 6.

When hemithioacetal protective groups are used, each sulfur atom to beprotected has a separate protective group attached to it, which togetherwith the sulfur atom defines a hemithioacetal group. The hemithioacetalgroups contain a carbon atom bonded directly (i.e., without anyintervening atoms) to a sulfur atom and an oxygen atom, i.e., ##STR8##

Preferred hemithioacetals generally are of the following formula,wherein the sulfur atom is a sulfur atom of the chelating compound, anda separate protecting group is attached to each of the sulfur atoms onthe chelating compound: ##STR9## wherein R³ is a lower alkyl group,preferably of from two to five carbon atoms, and R⁴ is a lower alkylgroup, preferably of from one to three carbon atoms. Alternatively, R³and R⁴ may be taken together with the carbon atom and the oxygen atomshown in the formula to define a nonaromatic ring, preferably comprisingfrom three to seven carbon atoms in addition to the carbon and oxygenatoms shown in the formula. R⁵ represents hydrogen or a lower alkylgroup wherein the alkyl group preferably is of from one to three carbonatoms. Examples of such preferred hemithioacetals include, but are notlimited to: ##STR10##

These sulfur-protective groups are displaced during the radiolabelingreaction, conducted at acidic pH, in what is believed to bemetal-assisted acid cleavage. Covalent bonds form between the sulfuratoms and the metal radionuclide. A separate step for removal of thesulfur-protective groups is not necessary. The radiolabeling procedurethus is simplified. In addition, the basic pH conditions and harshconditions associated with certain known radiolabeling procedures orprocedures for removal of other sulfur protective groups are avoided.Thus, base-sensitive groups on the chelating compound survive theradiolabeling step intact. Such base labile groups include any groupwhich may be destroyed, hydrolyzed, or otherwise adversely affected byexposure to basic pH. In general, such groups include esters,maleimides, and isothiocyanates, among others. Such groups may bepresent on the chelating compounds as protein, ligand or anti-ligandconjugation groups.

The compounds of the present invention preferably comprise at least one═O substituent, most preferably two ═O substituents. In one embodimentof the invention at least one and preferably two R₂ substituents are--(CH₂)_(n) --COOH, with n preferably equal to 1.

Examples of the chelating compounds of the present invention are thecompounds of the following formulas: ##STR11## wherein the symbols areas described above. Procedures for synthesizing these compounds arepresented in the examples below. In one embodiment of the invention,these chelating compounds comprise either two hemithioacetal, or onehemithioacetal and one acetamidomethyl sulfur protecting groups.

Other chelating compounds of the present invention incorporate one ormore saccharide residues. A preferred number of saccharide residuesranges from 1 to about 10, although when polymeric saccharides areemployed the number of saccharide residues therein may be higher.

Saccharides, such as hexoses (e.g., glucose) and pentoses (e.g.,fructose) and polymers of such saccharides are hydrophilic and,consequently, are generally excreted efficiently into the urine byglomular filtration. Inulin, a 5 kD polymer of fructose, is the goldstandard for glomular filtration studies. Derivatization of a chelatingcompound with one or more hexose residues, such as glucose residues, forexample, is expected to increase the water solubility and hydrophilicityof the chelating compound and conjugates containing the same.Consequently, glucose-bearing chelating compounds and conjugates willexhibit enhanced renal excretion.

Saccharide or saccharide derivative-bearing conjugates can be preparedin accordance with procedures discussed in the examples below forglucose derivative-bearing conjugates. Exemplary glucosederivative-containing conjugates of the present invention can beprepared from a variety of intermediates including glucose, glucosamine,gluconate, glucoheptonate, gluconic acid, gluconolactone, glucaric acid(i.e., saccharic acid), D-saccharic 1,4-lactone monohydrate, glucuronicacid, and the like. other sugars and sugar derivatives of similarfunctionalization are also commercially available and useful in thepractice of the present invention.

The choice of sugar derivative (unmodified, amino functionalized orcarboxy functionalized) depends on the conjugating group of thechelating compound. For example, amino sugars are preferred forconjugation to chelating compound carboxyl groups. Glucosamine, forexample, is useful for this purpose, as it allows amino group reactionwith appropriate derivatives of chelating compounds such as activeesters, active halides, aldehydes.

Alternatively, saccharide compounds bearing carboxyl residues arecommercially available and can be reacted with amine derivatives ofchelating compounds. Glucuronic acid, for example, is useful for thispurpose, as it bears a carboxy residue available for reaction with achelating compound amine.

Also, native saccharide compounds, such as glucose for example, can bereacted with chelating compound amines to form amine-linkedsugar-chelating compound conjugates. Subsequent or concurrent iminereduction results in a stable amine linkage.

Sugar lactones may be employed in the preparation of amide-linkedsugar-chelating compound conjugates. The lactone serves as an activatedcarboxylic acid which undergoes nucleophilic, ring opening upon reactionwith amine bearing chelating compounds.

The following table summarizes examples of saccharide (sugar)-chelatingcompound (non-sugar) chemical conjugates prepared vianucleophile-electrophile reaction:

    ______________________________________                                        Nucleophile                                                                           Electrophile   Sugar Derivative                                                                          Linkage                                    ______________________________________                                        sugar amine                                                                           activated carboxyl                                                                           glucosamine amide                                      amino   sugar aldehyde native sugar                                                                              amine                                      amino   activated sugar carboxyl                                                                     glucuronic acid                                                                           amide                                      amino   sugar lactone  gluconolactone                                                                            amide                                      hydrazide                                                                             sugar aldehyde native sugar                                                                              hydrazide                                  ______________________________________                                    

The chelating compounds of the present invention are radiolabeled, usingconventional procedures, with any of a variety of radionuclide metals toform the corresponding radionuclide metal chelates. These radionuclidemetals include, but are not limited to, copper (e.g., ⁶⁷ Cu and ⁶⁴ Cu);technetium (e.g., ^(99m) Tc); rhenium (e.g., ¹⁸⁶ Re and ¹⁸⁸ Re); lead(e.g., ²¹² Pb); bismuth (e.g, ²¹² Bi); and palladium (e.g., ¹⁰⁹ Pd).Methods for preparing these isotopes are known. Molybdenum/technetiumgenerators for producing ^(99m) Tc are commercially available.Procedures for producing ¹⁸⁶ Re include the procedures described byDeutsch et al., (Nucl. Med. Biol., Vol. 13:4:465-477, 1986) andVanderheyden et al. (Inorganic Chemistry, Vol. 24:1666-1673, 1985), andmethods for production of ¹⁸⁸ Re have been described by Blachot et al.(Intl. J. of Applied Radiation and Isotopes, Vol. 20:467-470, 1969) andby Klofutar et al. (J. of Radioanalytical Chem., Vol. 5:3-10, 1970).Production of ²¹² Pd is described in Fawwaz et al., J. Nucl. Med.(1984), 25:796. Production of ²¹² Pb and ²¹² Bi is described in Gansowet al., Amer. Chem. Soc. Symp. Ser. (1984), 241:215-217, and Kozah etal., Proc. Nat'l. Acad. Sci. USA (January 1986), 83:474-478. ^(99m) Tcis preferred for diagnostic use, and the other radionuclides listedabove have therapeutic use.

In one embodiment of the present invention, chelating compounds of theinvention comprising acetamidomethyl and/or hemithioacetal sulfurprotective groups are radiolabeled with a metal radionuclide by reactingthe compound with the radionuclide under conditions of acidic pH. It isbelieved that the acidic pH and the presence of the metal bothcontribute to the displacement of the sulfur protective groups from thechelating compound. The radionuclide is in chelatable form when reactedwith the chelating compounds of the invention.

In the case of technetium and rhenium, being in "chelatable form"generally requires a reducing step. A reducing agent will be employed toreduce the radionuclides (e.g., in the form of pertechnetate andperrhenate, respectively) to a lower oxidation state at which chelationwill occur. Many suitable reducing agents, and the use thereof, areknown. (See, for example, U.S. Pat. Nos. 4,440,738; 4,434,151; and4,652,440.) Such reducing agents include, but are not limited to,stannous ion (e.g., in the form of stannous salts such as stannouschloride or stannous fluoride), metallic tin, ferrous ion (e.g., in theform of ferrous salts such as ferrous chloride, ferrous sulfate, orferrous ascorbate) and many others. Sodium pertechnetate (i.e., ^(99m)TcO₄ ⁻ which is in the +7 oxidation level) or sodium perrhenate (i.e.,¹⁸⁸ ReO₄ ⁻, ¹⁸⁶ ReO₄ ⁻) may be combined simultaneously with a reducingagent and a chelating compound of the invention in accordance with theradiolabeling method of the invention, to form a chelate.

Preferably, the radionuclide is treated with a reducing agent and acomplexing agent to form an intermediate complex (i.e., an "exchangecomplex"). Complexing agents are compounds which bind the radionuclidemore weakly than do the chelate compounds of the invention, and may beweak chelators. Any of the suitable known complexing agents may be used,including but not limited to gluconic acid, glucoheptonic acid,methylene disphosphonate, glyceric acid, glycolic acid, mannitol, oxalicacid, malonic acid, succinic acid, bicine, N,N'-bis(2-hydroxy ethyl)ethylene diamine, citric acid, ascorbic acid and gentisic acid. Goodresults are obtained using gluconic acid or glucoheptonic acid as theTc-complexing agent and citric acid for rhenium. When the radionuclidein the form of such an exchange complex is reacted with the chelatingcompounds of the invention, the radionuclide will transfer to thesecompounds which bind the radionuclide more strongly to form chelates ofthe invention. Heating is often required to promote transfer of theradionuclide. Radionuclides in the form of such complexes also areconsidered to be in "chelatable form" for the purposes of the presentinvention.

Chelates of ²¹² Pb, ²¹² Bi, ¹⁰⁹ Pd may be prepared by combining theappropriate salt of the radionuclide with the chelating compound andincubating the reaction mixture at room temperature or at highertemperatures. It is not necessary to treat the lead, bismuth, palladium,and copper radioisotopes with a reducing agent prior to chelation, assuch isotopes are already in an oxidation state suitable for chelation(i.e., in chelatable form). The specific radiolabeling reactionconditions may vary somewhat according to the particular radionuclideand chelating compound involved.

The chelating compound may be radiolabeled to form a radionuclide metalchelate, which then is reacted with a targeting protein, ligand oranti-ligand. Alternatively, the unlabeled chelating compound may beattached to the targeting protein, ligand or anti-ligand andsubsequently radiolabeled. Proteins and proteinaceous ligands oranti-ligands (e.g., avidin or streptavidin) as well as non-proteinaceousligands or anti-ligands (e.g., biotin) contain one or more of a varietyof functional groups; e.g., carboxylic acid (COOH) or free amine (--NH₂)groups, which are available for reaction with a suitable protein, ligandor anti-ligand conjugation group "Z" on a chelator to bind the chelatorto the protein, ligand or anti-ligand. For example, an active ester onthe chelator reacts with primary amine groups on lysine residues ofproteins to form amide bonds. Alternatively, the protein, ligand oranti-ligand and/or chelator may be derivatized to expose or attachadditional reactive functional groups. The derivatization may involveattachment of any of a number of linker molecules such as thoseavailable from Pierce Chemical Company, Rockford, Ill. (See the Pierce1986-87 General Catalog, pages 313-54.) Alternatively, thederivatization may involve chemical treatment of the protein (which maybe an antibody), ligand or anti-ligand. Procedures for generation offree sulfhydryl groups on antibodies or antibody fragments are alsoknown. (See U.S. Pat. No. 4,659,839.) Maleimide conjugation groups on achelator are reactive with the sulfhydryl (thiol) groups.

Alternatively, when the targeting compound is a carbohydrate orglycoprotein, derivatization may involve chemical treatment of thecarbohydrate; e.g., glycol cleavage of the sugar moiety of aglycoprotein antibody with periodate to generate free aldehyde groups.The free aldehyde groups on the antibody may be reacted with free amineor hydrazine conjugation groups on the chelator to bind the chelatorthereto. (See U.S. Pat. No. 4,671,958.)

Biotin has a terminal carboxy moiety which may be reacted with asuitable ligand conjugation group, such as an amine, hydroxyl in thepresence of a coupling agent such as DCC or the like. In addition, theterminal carboxy moiety may be derivatized to form an active ester,which is suitable for reaction with a suitable ligand conjugation group,such as an amine, a hydroxyl, another nucleophile, or the like.Alternatively, the terminal carboxy moiety may be reduced to a hydroxymoiety for reaction with a suitable ligand conjugation group, such as ahalide (e.g., iodide, bromide or chloride), toxylate, mesylate, othergood leaving groups or the like. The hydroxy moiety may be chemicallymodified to form an amine moiety, which may be reacted with a suitableligand conjugation group, such as an active ester or the like.

The radiolabeled targeting proteins, ligands and anti-ligands of thepresent invention have use in diagnostic and therapeutic procedures,both for in vitro assays and for in vivo medical procedures. One type oftherapeutic or diagnostic procedure in which the compounds of thepresent invention may be employed is a pretargeting protocol. Generally,pretargeting encompasses two protocols, termed the three-step and thetwo-step. In the three-step protocol, shown schematically below,targeting moiety-ligand is administered and permitted to localize totarget. ##STR12## Targeting moiety-ligand conjugates may be prepared inaccordance with known techniques therefor. Anti-ligand is thenadministered to act as a clearing agent and to facilitate and direct theexcretion of circulating targeting moiety-ligand. The anti-ligand alsobinds to target-associated targeting moiety-ligand. Next, a conjugateemploying a compound of the present invention is administered, havingthe following structure:

    Ligand--------Chelate--------Radionuclide

The radiolabeled ligand conjugate either binds to target-associatedtargeting moiety-ligand-anti-14-gand or is rapidly excreted, with theexcretion proceeding primarily through the renal pathway. Consequently,the target-non-target ratio of active agent is improved, and undesirablehepatobiliary excretion and intestinal uptake of the active agent aresubstantially decreased.

Two-step pretargeting involves administration of targetingmoiety-anti-ligand, which may be prepared in accordance with knowntechniques therefor. After permitting the administered agent to localizeto target, a radiolabeled ligand of the present invention isadministered. Preferably, as a "step 1.5," a clearing agent isadministered to remove circulating targeting moiety-anti-ligand withoutbinding of clearing agent to target-associated targetingmoiety-anti-ligand. In this manner, the target-non-target ratio of theradiolabeled ligand is increased, and undesirable hepatobiliaryexcretion and intestinal uptake of the radiolabeled ligand aresubstantially decreased.

The radiolabeled proteins, ligands or anti-ligands may be administeredintravenously, intraperitoneally, intralymphatically, locally, or byother suitable means, depending on such factors as the type of targetsite. The amount to be administered will vary according to such factorsas the type of radionuclide (e.g., whether it is a diagnostic ortherapeutic radionuclide), the route of administration, the type oftarget site(s), the affinity of the targeting protein for the targetsite of interest, the affinity of the ligand and anti-ligand for eachother and any cross-reactivity of the targeting protein, ligand oranti-ligand with normal tissues. Appropriate dosages may be establishedby conventional procedures and a physician skilled in the field to whichthis invention pertains will be able to determine a suitable dosage fora patient. A diagnostically effective dose for a chelate labeledantibody embodiment of the present invention is generally from about 5to about 35 and typically from about 10 to about 30 mCi per 70 kg bodyweight. A therapeutically effective dose is generally from about 20 mCito about 300 mCi. Elevated doses, e.g., ranging from about 2 to about 10times higher, can be used when pretargeting procedures are employed,because of the decoupling of targeting moiety localization andradionuclide localization. For diagnosis, conventional non-invasiveprocedures (e.g., gamma cameras) are used to detect the biodistributionof the diagnostic radionuclide, thereby determining the presence orabsence of the target sites of interest (e.g., tumors).

The comparatively low intestinal localization of the therapeuticradiolabeled antibodies of the present invention or catabolites thereofpermits increased dosages, since intestinal tissues are exposed to lessradiation. The clarity and accuracy of diagnostic images also isimproved by the reduced localization of radiolabeled antibodies orcatabolites thereof in normal tissues. These advantages are alsoexperienced in the practice of the pretargeting aspects of the presentinvention.

The following examples are presented to illustrate certain embodimentsof the present invention.

EXAMPLE I Synthesis of S-acetamidomethyl-N-t-BOC IsocysteineTrichloroethyl Ester

The synthesis procedure is outlined in FIG. 1. Preparation ofS-acetamidomethyl-N-t-BOC isocysteine 6 from 1:

Mercaptosuccinic acid 1 (commercially available) was reacted withcyclopentanone in TosOH to form 2-oxathiolone 2.

To a solution of 2-oxathiolone 2 in benzene (40 mL) and triethylamine(3.28 mL, 23.55 mmol) at 0° C., was added a solution of diphenylphosphorylazide (5.08 mL, 23.55 mmol) in benzene (5.0 mL). The ice bathwas removed and the reaction solution was stirred at room temperaturefor 1 hour. The solution was washed with water. The water was extractedwith benzene. The combined benzene extracts were dried, concentrated tohalf the original volume, and heated under reflux in an oil bathgradually raised in temperature from 50° C. to 80° C. over 1 hour. Thereaction solution was cooled to room temperature, diluted with ethylacetate (50 mL) and washed twice with a saturated solution of NaHCO₃ (30mL). The organic extracts were dried (MgSO₄) and evaporated to give thecrude isocyanate 3 as a brown oil (4.92 g).

A suspension of 3 in 6N HCl (45 mL) was heated under reflux for 40minutes. The reaction solution was cooled, washed twice with ethylacetate (50 mL). Evaporation of the aqueous extract gave crudeisocysteine 4 as an amber oil (4.92 g, theoretical 3.64 g). NMR showsisocysteine plus an aliphatic contaminant.

To half of the crude isocysteine 4 (2.42 g, theoretical 11.61 mmol) inwater (3.0 mL) at 0° C. was added N-hydroxyacetamide (1.14 g). To thissolution was added dropwise concentrated HCl (0.45 mL). The solution wasstored at 0° C. for 3 days. The solution was evaporated to give S-acmisocysteine 5 as a colorless liquid NMR (D2O) 1.95 (s, 3H), 3.35 (dd,2H), 3.8 (t, 1H), 4.4 (dd, 2H). TLC (c-18, 15% meOH/H₂ O 1% HOAc, onespot 0.4 Rf.

To a solution of 5 (theoretical 11.61 mmol) in DMF/H₂ O 3:2, 25 mL) andtriethylamine (3.60 mL, 25-54 mmol) was added di-t-butyl dicarbonate(3.04 g, 13.9 mmol). The reaction was stirred at room temperature for 3hours and then evaporated. The residue was partitioned between water andethyl acetate. The water layer was acidified to pH 3.0 with 1.0M HCl andfurther extracted with ethyl acetate (3×30 mL) and methylene chloride(2×50 mL). The combined organic extracts were dried (MgSO₄) andevaporated to give an oil. Purification by chromatography (15%isopropanol/methylene chloride 2% acetic acid) afforded 6 as an oilwhich crystallized from acetonitrile. Yield from 2-oxathiolone 2 was1.90 g (6.21 mmol)=53%.

Conversion of S-acm N-T-BOC isocysteine 6 to S-acm N-T-BOC isocysteinetrichloroethyl ester 7:

To an ice cold solution of 6 (1.90 g, 6.21 mmol) and trichloroethanol(0.71 mL, 7.45 mmol) in acetonitrile (12 mL) and methylene chloride (2mL) was added dicyclohexylcarbodiimide (DCC) (1.47 g, 7.14 mmol) anddimethylamino pyridine (76 mg, 0.62 mmol). The ice bath was allowed tomelt and the reaction solution was stirred for 16 hours at roomtemperature. The reaction was cooled to 0° C., filtered, and evaporatedto give an oil which was purified by chromatography (1:1 EtOAc/Hexanes1% HOAc) to give 7 as an oil (1.25 g, 2.95 mmol) in 47% yield.

EXAMPLE II Synthesis of N-T-BOC Aminoadipic Acid δt-butyl Esterα-succinimidyl Ester 12

The synthesis procedure is outlined in FIG. 2. Conversion of N-t-BOCoxazolidine aminoadipic acid (9) to N-t-BOC oxazolidine aminoadipic acidt-butyl ester (10):

To an ice cold solution of 9 (3.23 g, 12.4 mmol) in acetonitrile (12 mL)and t-butanol (1.75 mL, 18.6 mmol) were added dimethylaminopyridine (151mg, 1.24 mmol) and DCC (3.07 g, 14.9 mmol). The reaction was stirred at0° C. for 69 minutes and then stored at 0° C. for 60 hours. The mixturewas filtered. The filtrate was evaporated to give a solid which waschromatographed (25% EtOAc/Hexanes). The t-butyl ester 10 was obtainedas a white solid (2.85 g, 8.66 mmol) in 70% yield. Conversion of 10 toN-t-BOC aminoadipic acid δ-t-butyl ester (11):

To a solution of 10 (100 mg, 0.30 mmol) in methanol (2.0 mL) was added1N NaOH (0.33 mL) dropwise. The solution was stirred for 1 hour and thentreated with ethanolamine (0.02 mL, 0.33 mmol). To this solution wasadded 1N NaOH (0.32 mL, 0.32 mmol). The reaction solution was stirredfor 48 hours, concentrated, and then neutralized by the addition of 1NHCl (0.33 mL). The aqueous phase was extracted with EtOAc (25 mL). Theaqueous phase was acidified with 1.0N HCl to pH 1 and further extractedwith EtOAc (2×50 mL). The combined EtOAc extracts were dried (MgSO₄),and evaporated to give an oil. Chromatography (40% EtOAc/Hexanes 1%HOAc) gave 11 as a colorless oil (60 mg, 0.19 mmol) in 63% yield.

Conversion of 11 to N-t-BOC aminoadipic acid δ-t-butyl esterα-succinimidyl ester 12

To an ice cold solution of 11 (0.97 g, 3.06 mmol) in acetonitrile (6.0mL) was added N-hydroxysuccinimide (422 mg, 3.67 mmol) and DCC (747 mg,3.67 mmol). The ice bath was allowed to melt and the reaction solutionwas stirred at room temperature for 5 hours. The mixture was cooled to0° C., treated with a few drops acetic acid, and filtered. Evaporationof the filtrate provided 12 as a white solid (1.19 g, 3.06 mmol) in 100%yield.

EXAMPLE III Synthesis of Succinate Reagent 16

Two procedures for synthesizing compound 16 are outlined in FIG. 3.

Procedure #1: Synthesis of succinate reagent 16 via base opening ofoxathiolone:

Conversion of 2 to 2-mercaptosuccinic acid oxathiolone β-t-butyl ester13

Compound 2 was prepared from 1 as described in Example I.

To an ice cold solution of 2 (1.45 g, 6.30 mmol) in acetonitrile (6.5mL) and t-butanol (0.89 mL) were added dimethyl aminopyridine (77 mg,0.63 mmol) and DCC 1.55 g, 7.56 mmol). The reaction was stirred for 1hour at 0° C. and then stored at 0° C. for 4 days. The product wasfiltered. The filtrate was evaporated. Chromatography (10%EtOAc/Hexanes) provided 13 as a yellow oil (1.76 g, 6.15 mmol) in 98%yield.

Conversion of 13 to 2-mercaptosuccinic acid β-t-butyl ester (14)

To a solution of 13 (0.58 g, 1.82 mmol) in acetone (2.0 mL) was added 1NNAOH (1.82 mL, 1.82 mmol). After the reaction solution was stirred for 4hours, additional 1N NaOH (1.82 mL, 1.82 mmol) was added. The reactionsolution was stirred for 20 hours, and then neutralized by the additionof 1.0M HCl (3.6 mL). The aqueous phase was extracted with EtOAc (3×25mL). The combined EtOAc extracts were washed with brine, dried andevaporated to give an oil. The product was chromatographed (first 10%EtOAc/Hexanes 10% HOAc, 300 mL, then 33% EtOAc/Hexanes 1% HOAc, 300 mL)to give 14 as colorless oil (0.24 g, 1.16 mmol) in 64% yield.

Conversion of 14 to S-tetrahydropyranylmercaptosuccinic acid β-t-butylester (15) and NHS ester 16

To a solution of 14 (240 mg, 1.16 mmol) and tosic acid monohydrate (7mg, 0.03 mmol) in methylene chloride at -40° C. was addeddihydro-2H-pyran (0.11 mL, 1.16 mmol). After the addition, the reactionwas warmed to -5° C. and stirred for 30 minutes. The solvent wasevaporated. The residue was dissolved in EtOAc (30 mL) and washed withpH 4.0 buffer. The aqueous phase was extracted with EtOAc (2×20 mL). Thecombined EtOAc extracts were washed with brine, dried and evaporated togive an oil which was used without purification. The oil was dissolvedin acetonitrile (2.0 mL), cooled to 0° C., and treated withN-hydroxysuccinimide (160 mg, 1.39 mmol) and DCC (287 mg, 1.39 mmol).The ice bath was allowed to melt and the reaction mixture was stirred atroom temperature for 20 hours. The mixture was filtered. The filtratewas evaporated. Chromatography provided 16 as a white solid (145 mg,0.37 mmol) in 32% yield.

Procedure #2: Synthesis of succinate reagent 16 using LDA

Conversion of S-tetrahydropyranylmercaptoacetic acid (17) toS-tetrahydropyranylmercaptosuccinic acid β-t-butyl ester 15 and NHSester 16

A solution of lithium diisopropylamide (LDA) was prepared by adding a1.30M solution of n-butyl lithium in hexanes (13.2 mL, 17.2 mmol) to asolution of diisopropyl amine (2.52 mL, 18.0 mmol) in THF (10.0 mL) at-78° C. The solution was stirred for 20 minutes. To this was addeddropwise a solution of S-tetrahydropyranylmercaptoacetic acid (1.32 g,7.50 mmol) in THF (5.0 mL). The reaction became cloudy. It was stirredat -78° C. for 25 minutes, warmed to 0° C., and stirred for 25 minutes.The reaction was then cooled to -78° C. and treated with a solution oft-butyl bromoacetate (3.2 mL) in THF (2.0 mL). The reaction solution wasstirred for 1 hour at -78° C., and for 30 minutes at 0° C. The reactionwas quenched by the addition of acetic acid (1.0 mL) in methylenechloride. The mixture was concentrated, diluted with water and ethylacetate. The aqueous layer was separated, acidified with 1.0M HCl to pH3.0, and further extracted with EtOAc (2×75 mL). The combined EtOAcextracts were washed with brine, dried, and evaporated to give 15 as acanary yellow oil.

The oil was dissolved in acetonitrile (10.0 mL) and methylene chloride(1.5 mL), cooled to 0° C., and treated with N-hydroxysuccinimide (1.03g, 9.0 mmol) and DCC (1.86 g, 9.0 mmol). The ice bath was allowed tomelt and the reaction mixture was stirred for 4 hours. The mixture wascooled to 0° C. and filtered. The filtrate was evaporated to give an oilwhich was chromatographed (305 EtOAc/Hexanes) to give 16 as a white foam(1.36 g, 3.51 mmol) in 47% yield.

EXAMPLE IV Synthesis of Isocys-aminoadipic-mercaptosuccinate ChelatingCompound 21

The synthesis procedure is outlined in FIG. 4. Condensation of cysteine8 with aminoadipic acid derivative 12 to give 17:

To an ice cold solution of S-acm N-T-BOC isocysteine trichloroethylester 7, prepared in Example I, (1008 mg, 2.38 mmol) in methylenechloride (7.0 mL) was added trifluoroacetic acid (6.0 mL) dropwise. Thesolution was stirred at room temperature for 1 hour. The solution wasevaporated from carbon tetrachloride (3×50 mL). The residue was dried invacuo for 18 hours. To an ice cold solution of the residue 8 in DMF (2.5niL) was added a solution of 12, prepared in Example II, (867 mg, 2.22mmol) in DMF (3.5 mL). To this was added triethylamine (0.73 mL, 5.24mmol). The reaction was stirred at room temperature for 6 hours and thenevaporated. The residue was partitioned between water and EtOAc. Theaqueous phase was extracted with EtOAc (2×50 mL). The combined EtOAcextracts were washed with brine, dried, and evaporated. The product waschromatographed (50% EtOAc/Hexanes 1% HOAc) to give 17 as a white foam(1005 mg, 1.61 mmol) in 68% yield.

Condensation of 17 with succinate reagent 16 to give tripeptide 18

To an ice cold solution 17 (500 mg, 0.81 mmol) in methylene chloride(4.3 mL) was added trifluoroacetic acid (4.3 mL). The ice bath wasremoved and the reaction was stirred for 1 hour. The solution wasevaporated from carbon tetrachloride (3×30 mL). The residue wasdissolved in DMF (1.0 mL) and cooled to 0° C. To this was added asolution of 16, prepared in Example III, (376 mg, 0.97 mmol) in DMF (2mL). Lastly triethylamine was added (0.22 mL, 1.62 mmol). The ice wasallowed to melt. The reaction was stirred at room temperature for 21hours. The solvent was evaporated. The residue was dissolved in EtOAcand washed with pH 4.0 buffer. The aqueous phase was extracted withEtOAc, then acidified with 1.0M HCl to pH 3.0. further extracted withEtOAc (2×30 mL). The combined EtOAc extracts were washed with brine,dried, and evaporated. The residue was chromatographed (99:1EtOAc:HOAc). The product 18 was obtained as a white solid in 80% yield(480 mg, 0.65 mmol).

Conversion of 18 to TFP ester 19

To an ice cold solution of 18 (480 mg, 0.65 mmol) in acetonitrile (1.5mL) and methylene chloride (0.5 mL) were added tetrafluorophenol (140mg, 0.84 mmol) and DCC (161 mg, 0.78 mmol). The ice bath was allowed tomelt and the reaction was stirred at room temperature for 20 hours. Thereaction was cooled to 0° C., treated with 2 drops acetic acid, andfiltered. The filtrate was evaporated. The residue was chromatographedto give 19 as an oil (240 mg, 0.27 mmol) in 42% yield.

Cleavage of TCE ester 19 to give 20

To a solution of 19 (190 mg, 0.21 mmol) in THF (1.4 mL) and 1.0M KH₂ PO₄(0.28 mL) was added Zn dust (137 mg, 2.10 mmol). The mixture was stirredfor 30 minutes. Additional phosphate buffer (0.28 mL) and Zn dust (137mg, 2.10 mmol) were added. The reaction was stirred for 80 minutes.Additional phosphate buffer (0.25 mL), THF (1.0 mL), and Zn dust (137mg, 2.10 mmol) were added. The reaction was filtered. The filtrate wasevaporated. The residue was chromatographed to give in the firstfractions recovered 19 (60 mg, 0.07 mmol), then in the later fractions20 as a white foam (40 mg, 0.05 mmol) in 25% yield.

Cleavage of t-butyl ester 20 to give 21

A solution of 20 (40 mg, 0.05 mmol) in formic acid (1.5 mL) was stirredfor 5 hours. The solution was evaporated. The product was purified bypreparative LC on reverse phase semi-prep C-18 column with 45% CH₃ CN/H₂O 1% HOAC as mobile phase. The product 21 was obtained as a film (6 mg,0.01 mmol) in 16% yield. The compound 21 is a chelating compound of thepresent invention.

EXAMPLE V Synthesis of Cysteine Monocarboxylate Chelating Compound 28

The synthesis procedure is outlined in FIG. 5. t-BOC cleavage andcondensation of cysteine 22 with aminoadipic acid derivative 12:

To an ice cold solution of 22 (0.97 g, 2.30 mmol) in methylene chloride(6.0 mL) was added trifluoroacetic acid (6.0 mL). The reaction wasstirred at room temperature, then coevaporated with carbon tetrachloride(3×15 mL) and dried in vacuo. The residue (23) was dissolved in dimethylformamide (1.0 mL) and triethylamine (0.35 mL, 2.53 mmol). To this wasadded a suspension of N-t-BOC aminoadipic acid-α-NHS-δ-t-butyl ester 12,prepared in Example II, (897 mg, 2.30 mmol) in DMF (2.5 mL).Triethylamine (0.35 mL, 2.53 mmol) was added and the reaction wasstirred for 18 hours. The solution was concentrated. The residue wasdissolved in EtOAc and washed with pH 4.0 buffer. The aqueous phase wasfurther extracted with EtOAc (2×30 mL). The combined EtOAc extracts werewashed with brine, dried, and evaporated to give an oil. Chromatography(75% EtOAc/Hexanes 1% HOAc) gave 24 as a white solid (1.40 g, 2.30 mmol)in 100% yield. FAB MS parent ions 622 and 624.

Deprotection of 24 and condensation with S-ethoxyethyl mercaptoaceticacid NHS ester to give 26

To an ice cold solution of 24 (690 mg, 1.12 mmol) in methylene chloride(6.0 mL) was added trifluoroacetic acid (6.0 mL). The ice bath wasremoved and the reaction was stirred at room temperature for 2 hours.The solution was coevaporated with carbon tetrachloride (3×10 mL). Theresidue was dissolved in DMF and triethylamine (0.15 mL, 1.12 mmol). Tothis solution at 0° C. was added a solution of S-ethoxyethylmercaptoacetic acid NHS ester (322 mg, 1.23 mmol) in DMF 2.0 mL). Lastlytriethylamine (0.31 mL, 2.24 mmol) was added. The ice bath was allowedto melt and the reaction was stirred at room temperature for 18 hours.The solvent was evaporated. The residue was dissolved in EtOAc (30 mL)and washed with pH 4.0 buffer. The aqueous phase was extracted withEtOAc (2×25 mL). The combined EtOAc extracts were dried and evaporated.The residue was chromatographed (50% EtOAc/Hexanes 1% HOAc). The product26 was obtained as an oil (380 mg, 0.55 mmol) in 50% yield.

Conversion of 26 to TFP ester 27

To a solution of 26 (190 mg, 0.31 mmol) in THF (1.8 mL) was addedtetrafluorophenol (65 mg, 0.35 mmol) and DCC (73 mg, 0.35 mmol). Thereaction was stirred for 20 hours, cooled to 0° C., and filtered. Thefiltrate was evaporated. The residue was chromatographed (99:1EtOAc:HOAc). The product 27 was obtained as colorless oil (150 mg, 0.20mmol) in 64% yield.

TCE ester cleavage of 27 to give cysteine ligand 28

To a solution of 27 (90 mg, 0.12 mmol) in THF (0-8 mL) and 1.0M KH₂ PO₄buffer (0.16 mL) was added Zn dust (78 mg, 1.20 mmol). The suspensionwas stirred for 40 minutes. Additional phosphate buffer (0.16 mL) and Zndust (78 mg, 1.20 mmol) were added. The reaction was stirred for 40minutes, filtered, and rinsed with 50% aqueous acetonitrile (30 mL). Thefiltrate was evaporated. The residue was chromatographed (15%isopropanol/methylene chloride 2% HOAc). The product 28 was obtained asan oil (60 mg, 0.10 mmol) in 80% yield. Compound 28 is a chelatingcompound of the present invention.

EXAMPLE VI Synthesis of Cysteine Succinate Chelating Compound 32

The synthesis procedure is outlined in FIG. 6. t-BOC and t-butylcleavage of 24 and condensation with succinate reagent 16 to giveprotected tripeptide 29:

To an ice cold solution of 24, prepared as in Example V, (708 mg, 1.16mmol) in methylene chloride (6.2 mL) was added trifluoroacetic acid (6.2mL). The solution was stirred at room temperature for 1.5 hours and thenevaporated from carbon tetrachloride (3×15 mL). To the residue dissolvedin DMF (2.0 mL) at 0° C. was added a solution of 16, prepared in ExampleIII, (450 mg, 1.16 mmol) in DMF (2.0 mL). The reaction was stirred for18 hours, and concentrated. The residue was partitioned between EtOAcand pH 4.0 buffer. The aqueous phase was extracted with EtOAc (2×25 mL).The combined EtoAc extracts were washed with brine, dried, andevaporated to give an oil. Chromatography (99:1 EtOAc/HOAc) provided 29as a white foam (0.39 g, 0.53 mmol) in 46% yield.

Conversion of 29 to TFP ester 30

To an ice cold solution of 29 (390 mg, 0.53 mmol) in acetonitrile (1.0mL) were added tetrafluorophenol (115 mg, 0.69 mmol) and DCC (131 mg,0.63 mmol) The reaction was stirred for 18 hours, cooled to 0° C.,filtered, and the filtrate was evaporated. Chromatography (75%EtOAc/Hexanes 1%; HOAc) gave 30 as an oil (400 mg, 0.45 mmol) in 85%yield.

Cleavage of t-butyl and trichloroethylester protecting groups to give 32

A solution of 30 (200 mg, 0.22 mmol) in formic acid (7.5 mL) was stirredfor 3 hours and then evaporated. The residue was chromatographed (99:1,EtOAclHOAc) to give 31 as a white foam. To a solution of 31 (180 mg,0.22 mmol) in THF (1.44 mL) were added Zn (144 mg, 2.20 mmol) and 1.0MKH₂ PO₄ (0.29 mL). The reaction was stirred 40 minutes. Additional Zn(150 mg, 2.29 mmol) and 1.0M KH₂ PO₄ (0.29 mL) were added. The reactionwas stirred for 30 minutes. Additional Zn (150 mg, 2.29 mmol) and 1.0MKH₂ PO₄ (0.29 mL) were added. The reaction was stirred 20 minutes,filtered, rinsed with acetonitrile (25 mL), 50% aqueous acetonitrile (10mL), and evaporated to give a solid (140 mg). One third of the crudeproduct was purified by preparative LC on a semi-analytical C-18 reverseLC column with 45% acetonitrile/water 1% acetic acid as the mobilephase. The final chelating compound 32 was obtained as a white film (17mg, 0.025 mmol). Thus projected yield if all of the crude product hadbeen LC prepped is 34% for the two deprotection steps. Compound 32 is achelating compound of the present invention.

EXAMPLE VII Synthesis of DAP-disuccinate 36

The synthesis procedure is outlined in FIG. 7. Condensation of 4,5-diaminopentanoic acid (DAP) with succinate reagent 16:

To an ice cold suspension of DAP (338 mg, 1.65 mmol) and 16, prepared inExample III, (1160 mg, 3.0 mmol) in DMF (3.5 mL) was added triethylamine(1.03 mL, 5.77 mmol). The ice bath was allowed to melt and the reactionwas stirred at room temperature for 18 hours. The solution wasconcentrated. The residue was partitioned between EtOAc and pH 4.0buffer. The aqueous phase was washed with EtOAc (2×50 mL). The combinedEtOAc extracts were washed with brine, dried, and evaporated. Theresidue was chromatographed (50% EtOAc/Hexanes 1% HOAc, 400 mL, then 65%EtOAc/Hexanes 1% HOAc) to give 34 as a white solid (770 mg, 1.13 mmol)in 69% yield.

Conversion of 34 to TFP ester 35

To an ice cold solution of 34 (363 mg, 0.50 mmol) in acetonitrile (1.0mL) and methylene chloride (0.1 mL) were added tetrafluorophenol (113mg, 0.68 mmol) and DCC (129 mg, 0.62 mmol). The ice bath was allowed tomelt and the reaction was stirred at room temperature for 18 hours. Thereaction was cooled to 0° C., treated with 2 drops acetic acid, andfiltered. The filtrated was evaporated. The residue was chromatographed(30% EtOAc/Hexanes) to give 35 as a white foam (350 mg, 0.41 mmol) in80% yield.

Conversion of 35 to discuccinate ligand 36

A solution of 35 (230 mg, 0.27 mmol) was stirred for 2 hours. Thesolution was coevaporated with toluene and dried in vacuo. Crude 36 wasobtained as a white solid (200 mg). Half of the product was purified bypreparative LC on a C-18 semi-prep reverse phase column. The firsteluting major peak, referred to as "A", was obtained in 22% yield as awhite solid (19 mg, 0.03 mmol). The second eluting major peak, referredto as "B" was obtained in 3906 yield (30 mg, 0.05 mmol). High resolutionFAB-MS showed parent ions and similar fragmentation patterns for bothisomers "A" and "B". Compound 36 (both isomers thereof) is a chelatingcompound of the present invention.

EXAMPLE VIII Preparation of Radionuclide Metal Chelates and Attachmentof the Chelates to Targeting Proteins

1. 99mTc Chelates: Each of the four chelating compounds synthesized inExamples I-VII (Compounds 21, 28, 32, and 36) was radiolabeled with^(99m) Tc according to the following procedure

One mL of sterile water for injection was added to a sterile vialcontaining a stannous gluconate complex (50 mg sodium gluconate and 1.2mg stannous chloride dehydrate, available from Merck Frosst, Canada, indry solid form) and the vial was gently agitated until the contents weredissolved. A sterile insulin syringe was used to inject 0.1 mL of theresulting stannous gluconate solution into an empty sterile vial. Sodiumpertechnetate (0.75 mL, 75-100 mCi, eluted from a "Mo/"Tc generatorpurchased from DuPont, Mediphysics, Mallinckrodt or E. R. Squibb) wasadded, and the vial was agitated gently to mix the contents, thenincubated at room temperature for 10 minutes to form a ^(99m)Tc-gluconate complex.

In an alternative procedure for providing the ^(99m) Tc-gluconateexchange complex, the kit includes a vial containing a lyophilizedpreparation comprising 5 mg sodium gluconate, 0.12 mg stannous chloridedehydrate, about 0.1 mg gentisic acid as a stabilizer compound, andabout 20 mg lactose as a filler compound. The amount of gentisic acidmay vary, with the stabilizing effect generally increasing up to about0.1 mg. Interference with the desired reactions may occur when about 0.2mg or more gentisic acid is added. The amount of lactose also may vary,with amounts between 20 and 100 mg, for example, being effective inaiding lyophilization. Addition of stabilizer and a filler compound isespecially important when the vial contained these relatively smallamounts of sodium gluconate and stannous chloride (compared to thealternative embodiment above). One mL of sodium pertechnetate (about 100mCi) was added directly to the lyophilized preparation. The vial wasagitated gently to mix the contents, then incubated as described aboveto form the ^(99m) Tc-gluconate complex.

A separate vial containing 0.3 mg of a chelating agent in dry solid formwas prepared by dispensing a solution of 0.3 mg chelating agent inacetonitrile into the vial, then removing the solvent under N₂ gas. Tothis vial was then added 0.87 mL of 100% isopropyl alcohol, and the vialwas gently shaken for about 2 minutes to completely dissolve thechelating compound. Next, 0.58 mL of this solution of the chelatingagent was transferred to a vial containing 0.16 mL of glacial aceticacid/0.2N HCl (2:14), and the vial was gently agitated. Of thisacidified solution, 0.5 mL was transferred to the vial containing the^(99m) Tc-gluconate complex, prepared above. After gentle agitation tomix, the vial was incubated in a 75° C.±2° C. water bath for 15 minutes,then immediately transferred to a 0° C. ice bath for 2 minutes.

To a separate vial containing 10 mg of the Fab fragment of a monoclonalantibody in 0.5 mL of phosphate-buffered saline, was added 0.37 mL of1.0M sodium bicarbonate buffer, pH 10.0. The Fab fragment was generatedby treating the monoclonal antibody with papain according toconventional techniques. The monoclonal antibody, designated NR-LU-10,recognizes a pancarcinoma antigen. The vial was gently agitated. Othertargeting proteins may be substituted for the NR-LU-10 Fab fragment.

The vial containing the acidified solution of the ^(99m) Tc-labeledchelate (see above) was removed from the ice bath, 0.1 mL of the sodiumbicarbonate buffer was added, and the vial was agitated to mix.Immediately, the buffered antibody solution (above) was added, gentlyagitated to mix and incubated at room temperature for 20 minutes toallow conjugation of the radiolabeled chelate to the antibody.

A column containing an anion exchanger, either DEAE-Sephadex orQAE-Sephadex, was used to purify the conjugate. Thuncolumn was preparedunder aseptic conditions as follows. Five 1 mL QAE-Sephadex columns wereconnected end-to-end to form a single column. Alternatively, a single 5mL QAE-Sephadex column may he used. The column was washed with 5 mL of37 mM sodium phosphate buffer, pH 6.8. A 1.2μ filter (available fromMillipore) was attached to the column, and a 0.2μ filter was attached tothe 1.2μ filter. A 22-gauge sterile, nonpyrogenic needle was attached tothe 0.2μ filter.

The reaction mixture was drawn up into a 3 mL or 5 mL syringe, and anyair bubbles were removed from the solution. After removal of the needle,the syringe was connected to the QAE-Sephadex column on the end oppositethe filters. The needle cap was removed from the 22-gauge needleattached to the filter end of the column and the needle tip was insertedinto a sterile, nonpyrogenic test tube. Slowly, over 2 minutes, thereaction mixture was injected into the column. The eluant collected inthe test tube was discarded. The now empty syringe on top of the columnwas replaced with a 5 mL syringe containing 5 mL of 75 mM (0.45%) sodiumchloride solution (from which air bubbles had been removed). The needleat the other end of the column was inserted aseptically into a sterile,nonpyrogenic 10 mL serum vial. Slowly, over 2 minutes, the NaCl solutionwas injected into the column, and the eluent was collected in the serumvial.

The resulting radiolabeled antibody fragments may be represented asfollows: ##STR13## 2. ¹⁸⁸ Re Chelates

The same chelating compounds may be radiolabeled with ¹⁸⁸ Re by aprocedure similar to the ^(99m) Tc labeling procedure. Sodium perrhenateproduced from a W-188/Re-188 research scale generator is combined withcitric acid (a preferred complexing agent for ¹⁸⁸ Re), a reducing agent,and preferably gentisic acid and lactose. The resulting ¹⁸⁸ Re-citrateexchange complex is heated with the desired chelating compound, asabove. A C₁₈ reversed phase low pressure material (Baker C₁₈ cartridges)may be used to purify the ¹⁸⁸ Re-chelate. A monoclonal antibody orfragment thereof is reacted with the chelate in a buffered solution tobind the chelate thereto, as described for the ^(99m) Tc procedure. ASephadex G-25 column may be used to purify the radiolabeled antibody.

EXAMPLE IX

Biodistribution of the four ^(99m) Tc-labeled antibody fragmentsprepared in Example VIII was analyzed in a rat model. 100 μg of protein(about 0.5 mCi) was administered intravenously into Sprague-Dawley rats.Each of the four types of radiolabeled antibody fragments (i.e.,NR-LU-10 Fab fragments radiolabeled with one of the four differentchelating compounds) was injected into three rats. Biodistribution wasanalyzed at 6 hours post-injection by isolating intestines and kidneysand determining the mCi of injected radioactivity per gram of thesetissues, using a dose calibrator. The percentage of injected dose pergram of intestinal and kidney tissue was calculated and averaged to givethe mean value for each group of three animals.

The results were compared with data on intestinal localization ofradioactivity for radiolabeled antibody fragments of the followingformula I (wherein the fragments are labeled with an N₂ S₂ chelate thatlacks carboxylic acid substituents): ##STR14##

A reduction in intestinal localization of radioactivity was demonstratedfor each of the four radiolabeled antibody fragments of the presentinvention, compared to the radiolabeled fragment of formula (I).

EXAMPLE X Preparation of Radiolabeled Antibody Fragments

1. ^(99m) Tc Chelates: Chelating compounds 21 and 36 (synthesized inExamples IV and VII, respectively) were radiolabeled with ^(99m) Tcaccording to the following procedure (a preferred procedure for thesetwo chelating compounds)

One mL of sterile water for injection was added to a sterile vialcontaining a stannous gluconate complex (50 mg sodium gluconate and 1.2mg stannous chloride dehydrate, available from Merck Frosst, Canada, indry solid form) and the vial was gently agitated until the contents weredissolved. A sterile insulin syringe was used to inject 0.1 mL of theresulting stannous gluconate solution into an empty sterile vial. Sodiumpertechnetate (0.75 mL, 75-100 mCi, eluted from a ⁹⁹ Mo/⁹⁹ Tc generatorpurchased from DuPont, Mediphysics, Mallinckrodt or E. R. Squibb) wasadded, and the vial was agitated gently to mix the contents, thenincubated at room temperature for 10 minutes to form a ^(99m)Tc-gluconate complex.

A separate vial containing 0.3 mg of the chelating agent (21 or 36) indry solid form was prepared by dispensing a solution of 0.3 mg chelatingagent in acetonitrile into the vial, then removing the solvent under N₂gas. To this vial was then added 0.87 mL of 100% isopropyl alcohol, andthe vial was gently shaken for about 2 minutes to completely dissolvethe chelating compound. Next, 0.58 mL of this solution of the chelatingagent was transferred to a vial containing 0.16 mL of glacial aceticacid/0.2N HCl (2:14), and the vial was gently agitated. Of thisacidified solution, 0.5 mL was transferred to the vial containing the^(99m) Tc-gluconate complex, prepared above. After gentle agitation tomix, the vial was incubated in a 75° C.±2° C. water bath for 15 minutes,then immediately transferred to a 0° C. ice bath for 2 minutes.

For compound 36, and whenever the radiolabeling yield for compound 21was below 40%, the radiolabeled chelate was purified prior toconjugation to an antibody as follows. An SPE-C₁₈ extraction column (areversed phase column available from Baker) was conditioned by washingwith 2 mL of ethanol followed by 2 mL of sterile water. The reactionmixture was then loaded onto the top of the column. The column waswashed with 2 mL aliquots of 1% ethanol/0.01M phosphate (pH=7.0) 6-8times and dried for 10 minutes under vacuum. The ^(99m) Tc chelates werethen eluted using 0.5 mL of CH₃ CN for compound 21 and 1 mL of CH₃ CNfor compound 36. The CH₃ CN was evaporated under a stream of N₂ prior tothe conjugation with antibody.

The ^(99m) Tc chelates thus purified were attached to the Fab fragmentof a monoclonal antibody (designated NR-LU-10) as described in ExampleVIII. Other targeting proteins may be substituted for the NR-LU-10antibody fragment.

EXAMPLE XI Preparation of ^(99m) Tc Chelate Using Chelating Compound 32

Compound 32 (prepared in Example VI) was radiolabeled by the followingprocedure, which is preferred for this particular chelating compound:

One mL of NaTcO₄ (˜100 mCi) was added to a lyophilized preparationcontaining 5.0 mg of sodium gluconate, 0.12 mg of stannous chloridedehydrate, 0.1 mg of gentisic acid, and 20 mg of lactose (lyophilizationpH=3.5). After incubating the vial at room temperature for 2 minutes,0.1 mL of compound 32 (1 mg/mL in 90% isopropyl alcohol) was added. Then0.300 mL of isopropyl alcohol and 0.060 mL of 0.1N HCl were added. 2 ccof air was added into the vial and incubated at 75° C. for 15 minutes.The vial was then immediately transferred to a 0° C. ice bath for 2minutes.

The resulting ^(99m) Tc chelate was attached to an antibody fragment asdescribed in Example VIII. Other targeting proteins may be substitutedfor the antibody fragment.

EXAMPLE XII Radiolabeled Ligand Preparation

A. A synthesis scheme for a N₂ S₂ -biotin conjugate is shown below:##STR15##

Epsilon-BOC-lysine a (available from Bachem Inc.) is acylated withN-hydroxy succinimidyl-S-tetrahydropyranyl mercaptoacetate b (preparablein accordance with known procedures for protecting thiols as5-tetrahydropyranyl hemithioacetals, such as Greene et al., ProtectiveGroups in Organic Synthesis, 2nd ed., page 291, John Wiley & Sons, Inc.,New York, 1990, to give N-alpha-(S-tetrahydropyranylmercaptoacetyl)-N-epsilon-BOC-lysine c. The BOC group is cleaved with formicacid, and the resultant amine d is acylated with NHS-biotin to give e.The free carboxyl group of e is activated with NHS and EDCI to give f,which is then coupled to S-acetarnidomethyl-cysteine to give theresultant N₂ S₂ -biotin conjugate g.

B. Diaminopentanoic acid (DAP) core N₂ S₂ -biotin conjugates of thefollowing general formula are contemplated as embodiments of the presentinvention: ##STR16## wherein X is H (synthesized using a5-biotinam4-do-pentylamine reactant, wherein the reactant is availablefrom Pierce Chemical Company) or COOH (synthesized using biocytin as areactant, wherein the reactant is available from Sigma Chemical Company)and wherein Y is H (synthesized using bis-EOE-mercaptoacetyl-DAP as areactant, wherein the reactant is synthesizable by known procedures) orCH₂ COOH (synthesized using bis-THP-mercaptosuccinyl-DAP as a reactant).

A one step synthesis for such DAP core N₂ S₂ -biotin conjugates is shownbelow: ##STR17##

A suspension of N₂ S₂ -tetrafluorophenylester or thioester a andbiocytin b is heated at 100° C. for 10 minutes. The product is purifiedby C-18 flash chromatography to afford the N₂ S₂ -biotin amide product.The 5-biotinamidopentylamine and N₂ S₂ -tetrafluorophenyl ester reactionoccurs analogously.

C. In the preparation of a conjugate as shown below, the followingprocedure may be employed. ##STR18##

1. A serine succinate reagent as shown below was produced as follows:

(a) t-butyl, N-hydroxy-succinimidyl succinate (LG694-73): To an ice coldsolution of succinic acid mono-t-butyl ester (870 mg, 5.0 mmol) and NHS(630 mg, 5.5 mmol) in acetonitrile (7.0 mL) was added DCC (1130 mg, 5.5mmol). The reaction was allowed to warm to room temperature and stirredfor 4.5 hours. The reaction was cooled to 0° C., treated with 0.1 mLacetic acid, and filtered. The filtrate was evaporated to give a gummysolid (1280 mg, theoretical yield). ¹ H NMR (CDCl₃): 1.40 (s, 9H), 2.60(t, 2H), 2.80 (s, 4H), 2.90 (t, 2H).

(b) serinyl succinate (LG694-97): To an ice cold suspension of sodiumhydride (60 mg, 2.49 mmol) in DMF (1.0 mL), was added a solution ofN-BOC serine (170 mg, 0.83 mmol). The suspension was stirred for 30minutes and then treated with a solution of t-butyl,N-hydroxy-succinimidyl succinate (225 mg, 0.83 mmol) in DMF (1.0 mL).The suspension was warmed to room temperature and stirred for 16 hours.The reaction was quenched at 0° C. by the addition of a solution ofacetic acid (0.1 mL) in EtOAc (1.0 mL). The suspension was partitionedbetween EtOAc and pH 4.0 buffer. The aqueous was extracted with EtOAc(2×30 mL). The aqueous was acidified to pH 1.0 with 1.0M HCl and furtherextracted with EtOAc (30 mL). The combined EtOAc extracts were washedwith brine, dried, and evaporated to give an oil. Chromatographyafforded the product as a colorless oil (190 mg, 0.53 mmol, 53%). ¹ HNMR (CDCl₃): 1.40 (2 overlapping singlets, 18H), 2.55 (broad s, 4H),4.40-5.50 (m, 2H), 4.60 (broad s, 1H) 5.50 (broad d, 1H). MS m/e (relintensity): 362 (M+H, 13), 337 (22), 250 (52), 154 (100).

(c) serinyl succinate NHS ester (LG694-79): To an ice cold solution ofserinyl succinate (600 mg, 1.66 mmol) and NF-S (229 mg, 1.99 mmol) inacetonitrile (2.5 mL) was added DCC (394 mg, 1.91 mmol). The reactionwas warmed to room temperature and stirred for 2 hours. The reaction wascooled to 0° C., treated with acetic acid (0.1 mL), and filtered. Thefiltrate was evaporated to give the product as an oil (760 mg, 1.66mmol, theoretical yield). ¹ H NMR (CDCl₃): 1.45 (2 overlapping singlets,18H), 2.55-2.70 (m, 4H), 2.85 (s, 4H), 4.55 (dd, 2H), 5.05 (broad s,1H), 5.60 (broad d, 1H).

2. A cysteine-serine succinate reagent as shown below was produced asfollows:

(a) S-acm-N-tBOC cysteine TCE ester (JRW 443-14): DCC (794 mg, 3.85mmol) was added to a solution of S-acm-N-tDOC cysteine (966 mg, 3.50mmol) and N,N-dimethyl aminopyridine (47 mg, 0.385 mmol) intrichloroethanol (0.37 mL, 3.85 mmol) and acetonitrile (18 mL). Thesuspension was stirred at room temperature for 48 hours. The suspensionwas filtered. The filtrate was evaporated. The residue was dissolved inEtOAc (75 mL) and washed with saturated NaHCO₃ (2×50 mL). The EtOAc wasdried, evaporated to give an oil which was crystallized fromether/hexanes to give the product as a white solid (500 mg, 1.22 mmol,3506) ¹ H NMR (CDCl₃): 1.50 (s, 9H), 2.05 (s, 3H), 3.10 (dd, 2H),4.40-4.60 (m, 2H), 4.60-4.95 (overlapping dd and s, 3H), 5.70 (broad d,1H), 6.90 (broad s, 1H). M.P. 76°-77° C.

(b) cys-ser-succinate (LG 694-80): To an ice cold solution ofS-acm-N-tBOC cysteine TCE ester (772 mg, 1.66 mmol) in CH₂ Cl₂ (3.0 mL),was added trifluoroacetic acid (4.0 mL). The reaction was warmed to roomtemperature and stirred for 1 hour. The solution was coevaporated withcarbon tetrachloride (3×30 mL). The residue was dissolved in DMF (1.5mL), cooled to 0°, and treated with triethylamine (0.24 mL, 1.72 mmol).To this solution was added a solution of serinyl succinate NHS ester(760 mg, 1.66 mmol) in DMF (1.5 mL). To this was added triethylamine0.48 mL, 3.32 mmol). The reaction was warmed to room temperature andstirred for 16 hours. The solution was diluted with EtOAc (50 mL) andwashed with 0.1M HCl, brine, dried, and evaporated to give an oil (1.4g). The oil was chromatographed (50% EtOAc:Hexanes 1% HOAc, 700 mL, then75% EtCAc:Hexanes 1% HOAc, then 99:1 EtOAc:HOAc, 200 mL) to give theproduct as an oil (200 mg, 0.30 mmol, 18%). ¹ H NMR (CDCl₃): 1.50(overlapping singlets, 18H), 2.05 (s, 3H), 2.55 (broad s, 4H), 3.15 (dd,2H), 4.40-4.65 (m, 5H), 4.70-5.05 (overlapping broad s, dd 3H), 5.70(broad d, 1H), 6.85 (broad s, 1H), 7.75 (broad d, 1H). MS m/e (relintensity): 668 (M+2, 5), 666 (M, 5), 568 (11), 510 (19), 439 (25), 57(100).

Alternatively and preferably, cys-ser-succinate is prepared as set forthbelow.

(b') N-t-BOC-(O-t-butyldimethylsilyl) serine (LG762-21): To a solutionof N-BOC-serine (615 mg, 3.00 mmol) and imidazole (449 mg, 6.60 mmol) inDMF (10.0 mL), was added t-butyldimethylsilyl chloride (994 mg, 6.60mmol). The reaction solution was stirred at room temperature for 15hours and then concentrated. The residue was dissolved in EtOAc (50 mL)and washed with water (25 mL), brine (25 mL), and dried to give theproduct as a white foam (1.00 g, 3.0 mmol, theoretical yield). ¹ H NMR(DMSO): 0.05 (s, 4H), 0.90 (s, 9H), 1.40 (s, 9H), 3.80 (t, 2H), 4.10 (m,1H), 6.75 (m, 1H).

N-t-BOC-serine-(O-t-butyldimethylsilyl)-N-hydioxy succinimidate(LG762-22): To an ice cold solution ofN-t-BOC-serine-O-t-butyldimethylsilyl ether (1.03 g, 3.0 mmol) inacetonitrile (4.5 mL) was added NHS (414 mg, 3.60 mmol) followed by DCC(712 mg, 3.45 mmol). The solution was warmed to room temperature andstirred for 16 hours. The mixture was treated with acetic acid (0.10mL), cooled to 0°, and filtered. The filtrate was evaporated to give theproduct as a white foam (1.25 g, 3.0 mmol, theoretical yield). ¹ H NMR(DMSO): 0.05 (s, 4H), 0.90 (s, 9H), 1.40 (s, 9H), 3.830 (s, 4H), 3.90(m, 2H), 4.50 (m, 1H), 7.45 (d, 1H).

(S-acetamido-methyl)-(trichloroethyl)!-cysteinyl.-N-BOC-(O-t-butyldimethylsilyl)-serine(LG762-32): To a solution of S-acm-N-tBOC cysteine TCE ester (1.02 g,2.41 mmol) in CH₂ Cl₂ (5.6 mL) was added trifluoroacetic acid (5.6 mL).The solution was stirred at room temperature for 1 hour and thencoevaporated with CCl₄ (3×30 mL). The residue was dissolved in DMF. Tothis solution at 0° was added a solution ofN-t-BOC-serine-O-t-butyldimethylsilyl-succinimidyl ester (1.00 g, 2.41mmol) in DMF (5.0 mL). To this was added triethylamine (0.84 mL, 6.02mmol). The reaction solution was stirred at room temperature for 16hours. Additional triethylamine (0.20 mL) was added and the reaction wasstirred for 1 hour. The, solution was concentrated. The residue wasdissolved in EtOAc (40 mL) and washed with pH 4.0 buffer. The aqueouswas washed with EtOAc (30 mL). The combined EtOAc extracts were washedwith brine, dried, and evaporated to give an oil. The oil waschromatographed (1:1 EtOAc:Hexanes 1% HOAC) to give the product as awhite foam (0.89 g, 1.52 mmol, 63%). ¹ H NMR (DMSO): 0.05 (s, 5H), 0.80(s, 9H), 1.35 (s, 9H), 1.85 (s, 3H), 2.95 (ddd, 2H), 3.70 (m, 2H), 4.20(m, 3H), 4.60 (m, 1H), 4.85 (dd, 2H), 6.65 (broad d, 1H), 8.55 (m, 2H).

(S-acetamidomethyl)-trichloroethyl-cysteinyl-N-BOC-serine (LG7G2-28): Asolution of(S-acetamidomethyl)-(trichloroethyl)!-cysteinyl-N-BOC-(O-t-butyldimethylsilyl)-serine(200 mg, 0.34 mmol) in 3:1:1 HOAc:H₂ O:THF (1.3 mL) was stirred at roomtemperature for 60 hours. The solution was partitioned between EtOAc andwater. The aqueous was extracted with EtOAc (2×20 mL). The combinedEtOAc extracts were washed with brine, dried, and evaporated to give theproduct as an oil (175 mg, 0.34 mmol, theoretical yield). ¹ H NMR(CDCl₃): 1.40 (s, 9H), 1.95 (s, 3H), 3.05 (ddd, 2H), 3.65 (dd, 1H), 4.05(dd, 1H), 4.15-4.50 (m, 3H), 4.70 (dd, 2H), 4.90 (m, 1H), 5.75 (m, 1H),6.70 (m, 1H), 7.75 (m, 1H).

(S-acetamidomethyl)-(trichloroethyl)!cysteinyl-N-BOC-O-(mono-t-btyl)-succinate!-serine (LG G94-80): To an ice cold solutionof (S-acetamidomethyl)-trichloroethyl-cysteinyl-N-BOC-serine (90 mg,0.18 mmol), succinic acid mono-t-butyl ester (31 mg, 0.18 mmol), andDMAP (24 mg, 0.19 mmol) in THF (0.6 mL), was added DCC (43 mg, 0.21mmol). The solution was warmed to room temperature and stirred for 18hours. The mixture was filtered and rinsed with cold acetonitrile. Thefiltrate was evaporated. The residue was chromatographed (50%EtOAc:Hexanes 1% HOAc, 300 mL, then 75% EtOAc:Hexanes 1% HOAc, 300 mL)to give the product as an oil (60 mg, 0.09 mmol, 51%). ¹ H NMR (CDCl₃):1.50 (s, 18H), 2.05 (s, 3H), 2.60 (broad s, 4H), 3.10 (m, 2H), 4.35-4.60(m, 5H), 4.60-5.05 (overlapping dd and s, 3H), 5.70 (m, 1H), 6.75 (broads, 1H), 7.70 (broad d, 1H).

(c) (S-acetamidomethyl)-(trichloroethyl)!cyisteinyl-N-(S-2-ethoxyethyl)mercaptoacetyl!- O-succinyl!-serine (LG64)4-81): To anice cold solution of cys-ser-succinate (200 mg, 0.30 mmol) in CH₂ Cl₂(2.0 mL), was added trifluoroacetic acid (2.0 mL). The solution wasstirred at room temperature for 1 hour. The solution was coevaporatedwith carbon tetrachloride (3×30 mL). The residue was dissolved in DMF(0.4 μmL) and cooled to 0°. To this solution was added triethylamine (42μL, 0.30 mmol). To this was added S-ethoxyethyl mercaptoacetic acidsuccinimidyl ester (94 mg, 0.36 mmol) and triethylamine (84 μL, 0.60mmol). The ice bath was allowed to melt and the reaction was stirred atroom temperature for 18 hours. The solution was diluted with EtOAc (50mL) and washed with pH 4.0 buffer. The aqueous was extracted with EtOAc(25 mL). The combined EtOAc: extracts were washed with brine, dried, andevaporated to give an oil. The oil was chromatographed (23 mm column,1:1 EtOAc:Hexanes 1% HOAc, 400 mL, then 99:1 EtOAc:HOAc, 700 mL, andlastly 10% IPA:EtOAc 1% HOAc) to give the product as a foam (80 mg, 0.12mmol, 41%). ¹ H NMR:1.25 (t, 3H), 1.50 (d, 3H), 2.05 (s, 3H), 2.50-2.70(m, 4H), 3.30 (m, 2H), 3.40-4.30 (m, SH), 4.35-4.95 (m, 8H), 6.90 (broads, 1H), 7.55-7.65 (broad t, 1H), 7.90-8.05 (broad t, 1H). MS m/e: 678(M+Na), 610, 391, 149.

(d) (S-acetamidomethyl)-(trichloroethyl)!cysteinyl-N-(S-2-ethoxyethyl)mercaptoacetyl!- O-(tetraflucrophenyl) succinate)serine(LG694-82): To an ice cold solution of cysteine-ser-succ SEOE ma (55 mg,0.08 mmol) in THF (0.50 mL), were added tetrafluorophenol (18 mg, 0.11mmol) and DCC (19 mg, 0.10 mmol). The ice bath was removed and thesolution was stirred at room temperature for 18 hours. The mixture wascooled to 0°, treated with acetic acid (0.1 mL), and filtered. Thefiltrate was evaporated to give an oil. The oil was chromatographed (1:1EtOAc:Hexanes 1%, HOAc, 200 mL, then 99:1 EtOAc:HOAc, 200 mL) to givethe product as an oil (45 mg, 0.055 mmol, 70%). ¹ H NMR (CDCl₃):1.20-1.35 (m, 3H), 1.55 (d, 3H), 2.10 (s, 3H), 2.82 (t, 2H), 3.05-3.1S(m, 4H), 3.40 (m, 2H), 3.50-3.85 (m, 2H), 4.35-5.05 (m, 9H), 6.70 (broads, 1H), 6.90-7.15 (m, 1H), 6.90-7.15 (m, 1H), 7.70 (m, 1H), 8.20 (broads, 1H). MS m/e (rel intensity): 806 (M+2, 14), 804 (M, 12), 761 (64),759 (52), 735 (35), 733 (28), 689 (37), 687 (37), 663 (410 (50), 155(100).

(e) S-acetamidomethyl)cysteinyl-N- (S-2-ethoxy) mercaptoacetyl!-O-(tetrafluorophenyl)succinate-serine (LG694-685): To a solution of theTFP ester (30 mg, 0.037 mmol) in THF (0.4 mL) and 1.0M KH₂ PO₄ (80 μL)was added zinc dust (39 mg, 0.59 mmol). After 1 hour, additional 1.0MKH₂ PO₄ (80 μL) and zinc dust (39 mg, 0.59 mmol) were added. The mixturewas agitated in a sonicator for 2 hours. The mixture was filtered,rinsed with acetonitrile and 50% CH₃ CN/H₂ O 1% HOAc. The filtrate wasevaporated. The residue was chromatographed (10% IPA:CH₂ Cl₂ 1% HOAc, 50mL, then 25% IPA:CH₂ Cl₂ 2% HOAc) to give the product as a foam (14 mg,0.02 mmol, 57%). ¹ H NMR (CDCl₃): 1.25 (t, 3H), 1.60 (d, 3H), 2.05 (s,3H), 2.85 (t, 2H), 3.10 (m, 4H), 3.35 (m, 2H), 3.40-3.80 (m, 2H),4.30-5.00 (m, 9H), 6.65 (m, 1H), 6.90-7.10 (m, 1H), 7.70 (m, 1H), 8.20(broad s, 1H).

3. The cysteine-serine succinate reagent is then utilized as shown belowto form the conjugate product. ##STR19## A suspension of biocytin (Xamidopentylamine (X=H) and the cysteine-serine succinate reagent in DMSOis heated for 10 minutes. The product is purified by C-18chromatography. The trichloroethyl ester protecting group is cleavedwith zinc dust in 1.0M potassium-dihydrogen phosphate to afford the fmalproduct.

EXAMPLE XIII Preparation of Targeting Moiety-Ligand and TargetingMoiety-Anti-Ligand Conjugates

A. Preparation and Characterization of Bictinylated Antibody

Biotinylated NR-LU-10 was prepared according to either of the followingprocedures. The first procedure involved derivitization of antibody vialysine ε-amino groups. NR-LU-10 was radioiodinated at tyrosines usingchloramine T and either ¹²⁵ I or ¹³¹ I sodium iodide. The radioiodinatedantibody (5-10 mg/ml) was then biotinylated using biotinamido caproateNHS ester in carbonate buffer, pH 8.5, containing 5% DMSO, according tothe scheme below. ##STR20##

The impact of lysine biotinylation on antibody immunoreactivity wasexamined. As the molar offering of biotin: antibody increased from 5:1to 40:1, biotin incorporation increased as expected (measured using theHABA assay and pronase-digested product) (Table 1, below). Percent ofbiotinylated antibody immunoreactivity as compared to native antibodywas assessed in a limiting antigen ELISA assay. The immunoreactivitypercentagre dropped below 70% at a measured derivitization of 11.1:1;however, at this level of derivitization, no decrease was observed inantigen-positive cell binding (performed with LS-180 tumor cells atantigen excess). Subsequent experiments used antibody derivitized at abiotin:antibody ratio of 10:1.

                  TABLE 1                                                         ______________________________________                                        Effect of Lysine Biotinylation on Immunoreactivity                            Molar     Measured      Immunoassessment                                      Offering  Derivitization                                                                              (%)                                                   (Biotins/Ab)                                                                            (Biotins/Ab)  ELISA   Cell Binding                                  ______________________________________                                         5:1       3.4          86                                                    10:1       8.5          73      100                                           13:1      11.1          69      102                                           20:1      13.4          36      106                                           40:1      23.1          27                                                    ______________________________________                                    

Alternatively, NR-LU-10 was biotinylated using thiol groups generated byreduction of cysteines. Derivitization of thiol groups was hypothesizedto be less compromising to antibody immunoreactivity. NR-LU-10 wasradioiodinated using p-aryltin phenylate NHS ester (PIP-NHS) and either¹²⁵ I or ¹³¹ I sodium iodide. Radioiodinated NR-LU-10 was incubated with25 mM dithiothreitol and purified using size exclusion chromatography.The reduced antibody (containing free thiol groups) was then reactedwith a 10- to 100-fold molar excess of N-iodoacetyl-n'-biotinyl hexylenediamine in phosphate-buffered saline (PBS), pH 7.5, containing 5% DMSO(v/v).

                  TABLE 2                                                         ______________________________________                                        Effect of Thiol Biotinylation on Immunoreactivity                             Molar     Measured      Immunoassessment                                      Offering  Derivitization                                                                              (%)                                                   (Biotins/Ab)                                                                            (Biotins/Ab)  ELISA   Cell Binding                                  ______________________________________                                        10:1      4.7           114                                                   50:1      6.5           102     100                                           100:1     6.1            95     100                                           ______________________________________                                    

As shown in Table 2, at a 50:1 or greater biotin:antibody molaroffering, only 6 biotins per antibody were incorporated. No significantimpact on immunoreactivity was observed.

The lysine- and thiol-derivitized biotinylated antibodies ("antibody(lysine)" and "antibody (thiol)", respectively) were compared. Molecularsizing on size exclusion FPLC demonstrated that both biotinylationprotocols yielded monomolecular (monomeric) IgGs. Biotinylated antibody(lysine) had an apparent molecular weight of 160 kD, while biotinylatedantibody (thiol) had an apparent molecular weight of 180 kD. Reductionof endogenous sulfhydryls to thiol groups, followed by conjugation withbiotin, may produce a somewhat unfolded macromolecule. If so, theantibody (thiol) may display a larger hydrodynamic radius and exhibit anapparent increase in molecular weight by chromatographic analysis. Bothbiotinylated antibody species exhibited 98% specific binding toimmobilized avidin-agarose.

Further comparison of the biotinylated antibody species was performedusing non-reducing SDS-PAGE, using a 4% stacking gel and a 5% resolvinggel. Biotinylated samples were either radiolabeled or unlabeled and werecombined with either radiolabeled or unlabeled avidin or streptavidin.Samples were not boiled prior to SDS-PAGE analysis. The native antibodyand biotinylated antibody (lysine) showed similar migrations; thebiotinylated antibody (thiol) produced two species in the 50-75 kDrange. These species may represent two thiol-capped species. Under theseSDS-PAGE conditions, radiolabeled streptavidin migrates as a 60 kDtetramer. When 400 μg/ml radiolabeled streptavidin was combined with 50μg/ml biotinylated antibody (analogous to "sandwiching" conditions invivo), both antibody species formed large molecular weight complexes.However, only the biotinylated antibody (thiol)-streptavidin complexmoved from the stacking gel into the resolving gel, indicating adecreased molecular weight as compared to the biotinylated antibody(lysine)-streptavidin complex.

B. Blood Clearance of Biotinylated Antibody Species

Radioiodinated biotinylated NR-LU-10 (lysine or thiol) was intravenouslyadministered to non-tumored nude mice at a dose of 100 μg. At 24 hpost-administration of radioiodinated biotinylated NR-LU-10, mice wereintravenously injected with either saline or 400 μg of avidin. Withsaline administration, blood clearances for both biotinylated antibodyspecies were biphasic and similar to the clearance of native NR-LU-10antibody.

In the animals that received avidin intravenously at 24 h, thebiotinylated antibody (lysine) was cleared (to a level of 5% of injecteddose) within 15 min of avidin administration (avidin:biotin=10:1). Withthe biotinylated antibody (thiol), avidin administration (10:1 or 25:1)reduced the circulating antibody level to about 35% of injected doseafter two hours. Residual radiolabeled antibody activity in thecirculation after avidin administration was examined in vitro usingimmobilized biotin. This analysis revealed that 85% of the biotinylatedantibody was complexed with avidin. These data suggest that thebiotinylated antibody (thiol)-avidin complexes that were formed wereinsufficiently crosslinked to be cleared by the RES.

Blood clearance and biodistribution studies of biotinylated antibody(lysine) 2 h post-avidin or post-saline administration were performed.Avidin administration significantly reduced the level of biotinylatedantibody in the blood, and increased the level of biotinylated antibodyin the liver and spleen. Kidney levels of biotinylated antibody weresimilar.

C. Preparation of Biotinylated Antibody (Thiol) Through EndogenousAntibody Sulfhydryl Groups Or Sulfhydryl-Generating Compounds

Certain antibodies have available for reaction endogenous sulfhydrylgroups. If the antibody to be biotinylated contains endogenoussulfhydryl groups, such antibody is reacted withN-iodoacetyl-n'-biotinyl hexylene diamine. The availability of one ormore endogenous sulfhydryl groups obviates the need to expose theantibody to a reducing agent, such as DTT, which can have otherdetrimental effects on the biotinylated antibody.

Alternatively, one or more sulfhydryl groups are attached to a targetingmoiety through the use of chemical compounds or linkers that contain aterminal sulfhydryl group. An exemplary compound for this purpose isiminothiolane. As with endogenous sulfhydryl groups (discussed above),the detrimental effects of reducing agents on antibody are therebyavoided.

D. Targeting Moiety-Anti-Ligand Conjugate for Two-Step Pretargeting

1. Preparation of SMCC-derivitized streptavidin.

31 mg (0.48 μmol) streptavidin was dissolved in 9.0 ml PBS to prepare afinal solution at 3.5 mg/ml. The pH of the solution was adjusted to 8.5by addition of 0.9 ml of 0.5M borate buffer, pH 8.5. A DMSO solution ofSMCC (3.5 mg/ml) was prepared, and 477 μl (4.8 μmol) of this solutionwas added dropwise to the vortexing protein solution. After 30 minutesof stirring, the solution was purified by G-25 (PD-10, Pharmacia,Piscataway, N.J.) column chromatography to remove unreacted orhydrolyzed SMCC. The purified SMCC-derivitized streptavidin was isolated(28 mg, 1.67 mg/ml).

2. Preparation of DTT-reduced NR-LU-10. To 77 mg NR-LU-10 (0.42 μmol) in15.0 ml PBS was added 1.5 ml of 0.5M borate buffer, pH 8.5. A DTTsolution, at 400 mg/ml (165 μl) was added to the protein solution. Afterstirring at room temperature for 30 minutes, the reduced antibody waspurified by G-25 size exclusion chromatography. Purified DTT-reducedNR-LU-10 was obtained (74 mg, 2.17 mg/ml).

3. Conjugation of SMCC-streptavidin to EITT- reduced NR-LU-10.DTT-reduced NR-LU-10 (63 mg, 29 ml, 0.42 pmol) was diluted with 44.5 mlPBS. The solution of SMCC-streptavidin (28 mg, 17 ml, 0.42 μmol) wasadded rapidly to the stirring solution of NR-LU-10. Total proteinconcentration in the reaction mixture was 1.0 mg/ml. The progress of thereaction was monitored by HPLC (Zorbax® GF-250, available from MacMod).After approximately 45 minutes, the reaction was quenched by addingsolid sodium tetrathionate to a final concentration of 5 mM.

4. Purification of conjugate. For small scale reactions, monosubstitutedconjugate was obtained using HPLC Zorbax (preparative) size exclusionchromatography. The desired monosubstituted conjugate product eluted at14.0-14.5 min (3.0 ml/min flow rate), while unreacted NR-LU-10 eluted at14.5-15 min and unreacted derivitized streptavidin eluted at 19-20 min.

For larger scale conjugation reactions, monosubstituted adduct isisolatable using DEAE ion exchange chromatography. After concentrationof the crude conjugate mixture, free streptavidin was removed therefromby eluting the column with 2.5% xylitol in sodium borate buffer, pH 8.6.The bound unreacted antibody and desired conjugate were thensequentially eluted from the column using an increasing salt gradient in20 mM diethanolaniine adjusted to pH 8.6 with sodium hydroxide.

5. Characterization of Conjugate.

a. HPLC size exclusion was conducted as described above with respect tosmall scale purification.

b. SDS-PAGE analysis was performed using 5% polyacrylamide gels undernon-denaturing conditions. Conjugates to be evaluated were not boiled insample buffer containing SDS to avoid dissociation of streptavidin intoits 15 kD subunits. Two product bands were observed on the gel, whichcorrespond to the mono- and di- substituted conjugates.

c. Immunoreactivity was assessed, for example, by competitive bindingELISA as compared to free antibody. Values obtained were within 10% ofthose for the free antibody.

d. Biotin binding capacity was assessed, for example, by titrating aknown quantity of conjugate with p- I-125!iodobenzoylbiocytin.Saturation of the biotin binding sites was observed upon addition of 4equivalences of the labeled biocytin.

e. In vivo studies are useful to characterize the reaction product,which studies include, for example, serum clearance profiles, ability ofthe conjugate to target antigen-positive tumors, tumor retention of theconjugate over time and the ability of a biotinylated molecule to bindstreptavidin conjugate at the tumor. These data facilitate determinationthat the synthesis resulted in the formation of a 1:1streptavidin-NR-LU-10 whole antibody conjugate that exhibits bloodclearance properties similar to native NR-LU-10 whole antibody, andtumor uptake and retention properties at least equal to native NR-LU-10.

For example, FIG. 8 depicts the tumor uptake profile of theNR-LU-10-streptavidin conjugate (LU-10-StrAv) in comparison to a controlprofile of native NR-LU-10 whole antibody. LU-10-StrAv was radiolabeledon the streptavidin component only, giving a clear indication thatLU-10-StrAv localizes to target cells as efficiently as NR-LU-10 wholeantibody itself.

EXAMPLE XIV Three-Step Pretargeting

A patient has ovarian cancer. A monoclonal antibody (MAb) directed to anovarian cancer cell antigen is conjugated to biotin to form a MAb-biotinconjugate. The MAb-biotin conjugate is administered to the patient in anamount in excess of the maximum tolerated dose of conjugateadministrable in a targeted, chelate labeled molecule protocol and ispermitted to localize to target cancer cells for 24-48 hours. Next, anamount of avidin sufficient to clear non-targeted MAb-biotin conjugateand bind to the targeted biotin is administered. A biotin-radionuclidechelate conjugate of the type discussed in Example XII above isdispersed in a pharmaceutically acceptable diluent and administered tothe patient in a therapeutically effective dose. The biotin-radionuclidechelate conjugate localizes to the targeted MAb-biotin-avidin moiety oris removed from the patient via the renal pathway.

EXAMPLE XV Two-Step Pretargeting

A patient has colon cancer. A monoclonal antibody (MAb) directed to acolon cancer cell antigen is conjugated to streptavidin to form aMAb-streptavidin conjugate. The MAb-streptavidin conjugate isadministered to the patient in an amount in excess of the maximumtolerated dose of conjugate administrable in a targeted, chelate labeledmolecule protocol and is permitted to localize to target cancer cellsfor 24-48 hours. A biotin-radionuclide chelate conjugate of the typediscussed in Example XII above is dispersed in a pharmaceuticallyacceptable diluent and administered to the patient in a therapeuticallyeffective dose. The biotin-radionuclide chelate conjugate localizes tothe targeted MAb-streptavidin moiety or is removed from the patient viathe renal pathway.

EXAMPLE XVI Glucose-Bearing Conjugates

A. Amide-linked Conjugate. The N₂ S₂ -biotin final product of ExampleXII(A) above has one free carboxyl group located on the chelatingcompound portion thereof. This carboxyl group is converted to aN-hydroxysuccinimidyl group using NHS and DCC in DMF. Side product DCUis removed by filtration. The filtrate is evaporated, and the residueNHS ester is used without further purification.

The residue is dissolved in DMF and three equivalents of triethylamineis added. To this solution is added glucosamine hydrochloride(commercially available from Aldrich Chemical Co., Milwaukee, Wis.).Progress of the reaction is monitored by thin layer chromatography(TLC). When TLC shows that the reaction is complete, the DMF andtriethylamine are evaporated, and the product glucose-N₂ S₂ -biotin ispurified by preparative reverse phase HPLC.

B. Multiple-glucose Conjugate. The N₂ S₂ -biotin final product ofExample XII(B) above has two free carboxyl groups located on thechelating compound portion thereof. These carboxyl groups are free forreaction with a glucose-bearing moiety to form a glucose-bearingconjugate. The carboxyl groups are activated by conversion toN-hydroxysuccinimidyl esters using NHS and DCC in DMF. Side product DCUis removed by filtration. The filtrate is evaporated, and the residuebis-NHS ester is used without further purification.

The residue is dissolved in DMF containing six equivalents oftriethylamine. To this solution is added two equivalents of glucosaminehydrochloride (commercially available from Aldrich Chemical Co.,Milwaukee, Wis.). Progress of the reaction is monitored by TLC. When TLCshows that the reaction is complete, the DMF and triethylamine areevaporated, and the product glucose-N₂ S₂ -biotin is purified bypreparative reverse phase HPLC.

C. Amine-bearing Chelating Compound-Biotin Conjugate Synthesis. Anexemplary amine-bearing chelating compound synthesis proceeds through anepsilon-N-t-BOC-alpha-N-methyl-lysine intermediate. This intermediate iseither commercially available or preparable in accordance with thefollowing procedure.

1. Synthesis of epsilon-N-t-BOC-alpha-N-methyl lysine.Epsilon-N-t-BOC-lysine was mono-alpha-N-methylated by the followingthree step procedure. Epsilon-N-t-BOC-lysine was reacted withtrifluoroacetic anhydride in THF. The trifluoroacetamide product wasmethylated by stirring a suspension ofepsilon-N-t-BOC-alpha-N-TFA-lysine, sodium hydride and methyl iodide inDMF at room temperature for about 6 hours. Following isolation ofepsilon-N-t-DOC-alpha-N-methyl-alpha-N-TFA-lysine by aqueous/organicextraction, the TFA group was cleaved by stirring a solution of theTFA-lysine derivative in a solution of 1:1 piperidine/THF at roomtemperature for about 4 hours.

2. Synthesis of alpha-N-methyl-alpha-N-biotinyl-L-lysine. The aminogroup is biotinylated by stirring the epsilon-N-t-BOC-alpha-N-methyllysine with biotin-NHS ester in DMF at room temperature for 8 hours. TheBOC group is cleaved by stirring a solution ofepsilon-N-t-BOC-alpha-N-methyl-alpha-N-biotinyl lysine intrifluoroacetic acid at room temperature for about 1 hour to formalpha-N-methyl-alpha-N-biotinyl-L-lysine.

3. Synthesis of an N₂ S₂ chelating compound bearing a protected lysineepsilon amino group and an activated ester reactive group.Epsilon-N-t-BOC-lysine and S-tetrahydropyranyl mercaptoaceticacid-N-hydroxysuccinimidyl ester (prepared in accordance with proceduresset forth in U.S. Pat. No. 4,965,392, for example) are stirred at roomtemperature in DMF containing 2.0 equivalents triethylamine to givealpha-N- S-tetrahydropyranylmercapto-acetyl!-epsilon-N-t-BOC-lysine. Thefree carboxyl group of this intermediate is activated by stirring withN-hydroxysuccinimide and DCC to give the N-hydroxysuccinimidyl ester.The NHS ester and S-acetamiclomethylcysteine (commercially availablefrom Bachem Calif., Torrence, Calif.) are condensed in DMF containing2.0 equivalents triethylamine to give the diamide product, alpha-N-S-tetrahydropyranylmercaptoacetyl!-epsilon-N-t-DOC-lysyl-S-acetamidomethyl cysteine. The free carboxyl group of this intermediate isactivated with NFS and DCC to give an activated NHS ester product.

4. Synthesis of amine-bearing chelating compound-biotin conjugate. TheN-protected amine-bearing chelate compound activated ester formed inaccordance with Subsection C3 of this Example is condensed withalpha-N-methyl-alpha-N-biotinyl-L-lysine formed in accordance withSubsection C2 of this example to give the N-protected chelatingcompound-biotin conjugate, alpha-N-S-tetrahydropyranylmercaptoacetyl!-epsilon-N-t-BOC-lysyl-(S-acetamidomethyl)-cysteinyl-epsilon-N-alpha-N-methyl-alpha-N-biotinyl!-lysine. The BOC protecting group ofthis intermediate is cleaved with formic acid to give the product,alpha-N-S-tetrahydropyranylmercaptoacetyl!-lysyl-(S-acetamidomethyl)-cysteinyl-epsilon-N-(alpha-N-methyl-alpha-N-biotinyl)-lysine.

D. Amide-linked, Biotinylated, Sugar-derivatized Conjugate. Thechelating compound prepared in accordance with the procedure describedin Section C of this Example is conjugated to glucuronic acid(commercially available from Aldrich Chemical Co., Milwaukee, Wis.)using the water soluble coupling agent EDCI. A solution containingequilmolar amounts of the chelating compound, glucuronic acid, EDCI andtriethylamine in DMF is stirred for about 12 hours at room temperature,for example. The solvents are then evaporated. The product amine-linkedconjugate is purified by reverse phase C-18 chromatography.

What is claimed is:
 1. A compound of the formula: ##STR21## wherein:each R independently represents ═O, H₂, lower alkyl, --(CH₂)_(n) --COOH,--(CH₂)_(n) --CO-glucose or glucose derivative, --(CH₂)_(n) --NH-glucoseor glucose derivative, or R₁ --Z;n is 0 to 3; R₁ represents a loweralkyl or substituted lower alkyl group; Z represents biotin or biotinwith a linker moiety; each R₂ independently represents H₂, a lower alkylgroup, --(CH₂)_(n) --COOH, --(CH₂)_(n) --CO-glucose or glucosederivative, --(CH₂)_(n) --NH-glucose or glucose derivative, or R₁ --Z;each m is 0 or 1, wherein at most one m is m=1; each T represents asulfur protecting group; and the compound comprises at least one--(CH₂)_(n) --CO-glucose, --(CH₂)_(n) --NH-glucose or glucose derivativeand at least one --R₁ --Z substituent.
 2. The compound of claim 1wherein R₁ is a methylene chain comprising from two to three carbonatoms.
 3. The compound of claim 1 wherein two R substituents are ═O. 4.The compound of claim 1 wherein at least one R₂ substituent is--(CH₂)_(n) --COOH.
 5. The compound of claim 1 wherein at least one Trepresents a hemithioacetal sulfur protecting group.
 6. A compound ofthe formula: ##STR22## wherein: M represents a radionuclide metal or anoxide thereof;each R independently represents ═O, H₂, lower alkyl,--(CH₂)_(n) --COOH, --(CH₂)_(n) --CO-glucose or glucose derivative,--(CH₂)_(n) --NH-glucose or glucose derivative, or R₁ --Z; n is 0 to 3;R₁ represents a lower alkyl or substituted lower alkyl group; Zrepresents a ligand or an anti-ligand or a ligand with a linker moietyor an anti-ligand with a linker moiety; each R₂ independently representsH₂ lower alkyl, --(CH₂)_(n) --COOH, --(CH₂)_(n) --CO-glucose or glucosederivative, --(CH₂)_(n) --NH-glucose or glucose derivative, or R₁ --Z;each m is 0 or 1, wherein at most one m is m=1; and the compoundcomprises at least one --(CH₂)_(n) --CO-glucose, --(CH₂)_(n)--NH-glucose or glucose derivative substituent and at least one --R₁ --Zsubstituent.
 7. The compound of claim 6 wherein R₁ is a methylene chaincomprising from two to three carbon atoms.
 8. The compound of claim 6wherein two R substituents are ═O.
 9. The compound of claim 6 wherein atleast one R₂ substituent is --(CH₂)_(n) --COOH.
 10. The compound ofclaim 6 wherein M represents a radionuclide metal selected from thegroup consisting of ^(99m) Tc, ¹⁸⁶ Re, ¹⁸⁸ Re, and oxides thereof.
 11. Acompound of the formula: ##STR23## wherein: each R independentlyrepresents ═O, H₂, lower alkyl, --(CH₂)_(n) --COOH, --(CH₂)_(n)--CO-glucose or glucose derivative, --(CH₂)_(n) --NH-glucose or glucosederivative, or R₁ --Z;n is 0 to 3; R₁ represents a lower alkyl orsubstituted lower alkyl group; Z represents biotin or a biotinconjugation group or a biotin with a linker moiety; each R₂independently represents H₂, a lower alkyl group, --(CH₂)_(n) --COOH,--(CH₂)_(n) --CO-glucose or glucose derivative, --(CH₂)_(n) --NH-glucoseor glucose derivative, or R₁ --Z; each m is 0 or 1, wherein at most onem is m=1; each T represents a sulfur protecting group; and the compoundcomprises at least one --(CH₂)_(n) --COOH glucose, --(CH₂)_(n)--NH-glucose or glucose derivative and at least one --R₁ --Zsubstituent.
 12. A compound of the formula: ##STR24## wherein: each Trepresents a sulfur protecting group;each R independently represents ═O,H₂, lower alkyl, --(CH₂)_(n) --COOH, --(CH₂)_(n) --CO glucose or glucosederivative, --(CH₂)_(n) --NH-glucose or glucose derivative, or R₁ --Z; nis 0 to 3; R₁ represents a lower alkyl or substituted lower alkyl group;Z represents a ligand or an anti-ligand or a ligand with a linker moietyor an anti-ligand with a linker moiety; each R₂ independently representsH₂ lower alkyl, --(CH₂)_(n) --COOH, --(CH₂)_(n) --CO-glucose or glucosederivative, --(CH₂)_(n) --NH-glucose or glucose derivative, or R₁ --Z;each m is 0 or 1, wherein at most one m is m=1; and the compoundcomprises at least one --(CH₂)_(n) --CO-glucose, --(CH₂)_(n)--NH-glucose or glucose derivative substituent and at least one --R₁ --Zsubstituent.
 13. A compound according to claim 1, additionally includingM, wherein M represents a radionuclide metal or an oxide thereof.